Novel glycosyl hydrolase enzymes and uses thereof

ABSTRACT

The present disclosure is generally directed to glycosyl hydrolase enzymes, compositions comprising such enzymes, and methods of using the enzymes and compositions, for example for the saccharification of cellulosic and hemicellulosic materials into sugars.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/810434, filed Jul. 27, 2015, which is a continuation of U.S.application Ser. No. 13/498,069, filed Apr. 1, 2013, which is a U.S.National Stage Application of International Appl. No. PCT/US10/49849,filed Sep. 22, 2010, which claims priority to U.S. ProvisionalApplication Nos. 61/245,273, filed Sep. 23, 2009, and 61/289,886, filedDec. 23, 2009. All these previous applications are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS on Jul. 27, 2015, in compliancewith 37 C.F.R. § 1.52(e), is incorporated herein by reference. Thesequence listing text file contains the file“NB31376-US-PCD_Sequence-Listing.txt” created on Feb. 7, 2019, which is176 KB (180,752 bytes).

2. TECHNICAL FIELD

The present disclosure generally pertains to glycosyl hydrolase enzymes,compositions comprising such enzymes, and methods of using the enzymesand compositions, for example for the saccharification or conversion ofcellulosic and hemicellulosic materials into sugars.

3. BACKGROUND

Bioconversion of renewable lignocellulosic biomass to a fermentablesugar that is subsequently fermented to produce alcohol (e.g., ethanol)as an alternative to liquid fuels has attracted the intensive attentionof researchers since the1970s, when the oil crisis occurred because OPECdecreased the output of petroleum (Bungay, H. R., “Energy: the biomassoptions”. N.Y.: Wiley; 1981; Olsson L, Hahn-Hagerdal B. Enzyme MicrobTechnol 1996,18:312-31; Zaldivar, J et al., Appl Microbiol Biotechnol2001, 56: 17-34; Galbe, M et al., Appl Microbiol Biotechnol 2002,59:618-28). Ethanol has been widely used as a 10% blend to gasoline inthe USA or as a neat fuel for vehicles in Brazil in the last twodecades. The importance of fuel bioethanol will increase in parallelwith skyrocketing prices for oil and gradual depletion of its sources.Additionally, fermentable sugars are increasingly used to produceplastics, polymers and other biobased products, and this industry isexpected to expand substantially in the coming years. Thus, the demandfor abundant low cost fermentable sugars, which can be used as a feedstock in lieu of petroleum based feedstocks, continues to grow.

Chiefly among the useful renewable feedstocks are cellulose andhemicellulose (xylans), which can be converted into fermentable sugars.The enzymatic conversion of these polysaccharides to soluble sugars, forexample glucose, xylose, arabinose, galactose, mannose, and/or otherhexoses and pentoses, occurs due to combined actions of various enzymes.For example, endo-1,4-β-glucanases (EG) and exo-cellobiohydrolases (CBH)catalyze the hydrolysis of insoluble cellulose to cellooligosaccharides(e.g., with cellobiose being a main product), while β-glucosidases (BGL)catalyzes the conversion of the oligosaccharides to glucose. Xylanasestogether with other accessory proteins (non-limiting examples of whichinclude L-α-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis ofhemicelluloses.

The cell walls of plants are composed of a heterogeneous mixture ofcomplex polysaccharides that interact through covalent and noncovalentmeans. Complex polysaccharides of higher plant cell walls include, forexample, cellulose (β-1,4 glucan), which generally constitutes 35-50% ofcarbon found in cell wall components. Cellulose polymers self associatethrough hydrogen bonding, van der Waals interactions and hydrophobicinteractions to form semi-crystalline cellulose microfibrils. Thesemicrofibrils also include noncrystalline regions, generally known asamorphous cellulose. The cellulose microfibrils are embedded in a matrixformed of hemicelluloses (including, e.g., xylans, arabinans, andmannans), pectins (e.g., galacturonans and galactans), and various otherβ-1,3 and β-1,4 glucans. These matrix polymers are often substitutedwith, for example, arabinose, galactose and/or xylose residues to yieldhighly complex arabinoxylans, arabinogalactans, galactomannans, andxyloglucans. The hemicellulose matrix is, in turn, surrounded bypolyphenolic lignin.

The complexity of the matrix makes it difficult to degrade bymicroorganisms as lignin and hemicellulose components must be brokendown before enzymes can act on the core cellulose microfibrils.Ordinarily, a consortium of different enzymatic activities is requiredto break down cell wall polymers to release the constituentmonosaccharides. For saccharification of plant cell walls, the ligninmust be permeabilized and hemicellulose disrupted to allowcellulose-degrading enzymes to act on their substrate.

Regardless of the type of cellulosic feedstock, the cost and hydrolyticefficiency of enzymes are major factors that restrict thecommercialization of biomass bioconversion processes. The productioncosts of microbially produced enzymes are tightly connected with theproductivity of the enzyme-producing strain and the final activity yieldin the fermentation broth. The hydrolytic efficiency of a multienzymecomplex depends both on properties of individual enzymes, the synergiesamong them, and their ratio in the multienzyme blend.

There exists a need in the art to identify enzyme and/or enzymaticblends/compositions that are capable of converting plant and/or othercellulosic or hemicellulosic materials into fermentable sugars withimproved efficacy and yield.

4. SUMMARY

The disclosure provides certain glycosyl hydrolase polypeptides havinghemicellulolytic activity, including, e.g., xylanases (e.g.,endoxylanases), xylosidases (e.g., β-xylosidases), arabinofuranosidases(e.g., L-α-arabinofuranosidases), nucleic acids encoding thesepolypeptides, and methods for making and using the polypeptides and/ornucleic acids. The disclosure is based, in part, on the discovery ofnovel enzymes and variants having xylanase, β-xylosidase, and/orL-α-arabinofuranosidase activities. The disclosure is also based on theidentification of enzyme blends (or compositions) that efficientlycatalyze the hydrolysis of cellulosic and hemicellulosic materials. Forpurpose of this disclosure, an enzyme can be defined either as apolypeptide having the particular enzymatic activity or as that enzyme.For example, a xylanase can be referred to as a polypeptide havingxylanase activity or as a xylanase enzyme, and a β-xylosidase can bereferred to as either a polypeptide having β-xylosidase activity or aβ-xylosidase enzyme.

The enzymes and/or enzyme blends/compositions of the disclosure can beused to produce sugars from biomass. The sugars so produced can be usedby microorganisms for ethanol production or can be used to produce otherbioproducts in various industrial applications. Therefore, thedisclosure also provides industrial applications (e.g., saccharificationprocesses in ethanol production) using the enzymes and/or enzymeblends/compositions described herein. The enzymes and/or enzymeblends/compositions of the present disclosure can be used to decreaseenzyme costs in biofuel production.

In one aspect, the invention of the disclosure pertains to enzymes(including variants thereof), or enzyme blends/compositions that areuseful for hydrolyzing the major components of a lignocellulosic biomass(including, e.g., cellulose, hemicellulose, and lignin) or any materialcomprising cellulose and/or hemicellulose. Such lignocellulosic biomassand/or material comprising cellulose and/or hemicellulose include, e.g.,seeds, grains, tubers, plant waste or byproducts of food processing orindustrial processing (e.g., stalks), corn (including, e.g., cobs,stover, and the like), grasses (e.g., Indian grass, such as Sorghastrumnutans; or, switchgrass, e.g., Panicum species, such as Panicumvirgatum), wood (including, e.g., wood chips, processing waste), paper,pulp, recycled paper (e.g., newspaper).

The enzyme blends/compositions of the invention can be used to hydrolyzecellulose comprising a linear chain of β-1,4-linked glucose moieties, orhemicellulose, of a complex structure that varies from plant to plant.

The enzyme blends/compositions of the invention can comprise a number ofdifferent enzymes, including, e.g., cellulases and/or hemicellulases.For example, the enzymes blends/compositions of the invention can beused to hydrolyze biomass or a suitable feedstock. The enzymeblends/compositions of the invention desirably comprise mixtures ofenzymes, selected from, e.g., xylanases, xylosidases,cellobiohydrolases, arabinofuranosidases, and/or other enzymes that candigest hemicellulose to monomer sugars. An enzyme blend/composition ofthe invention can comprise a mixture of two or more, three or more, orfour or more enzymes selected from one or more xylanases, one or morexylosidases, one or more cellobiohydrolases, one or morearabinofuranosidases, and one or more other enzymes that are capable ofconverting hemicelluose to monomer sugars. The other enzymes that candigest hemicellulose to monomer sugars include, without limitation, acellulase, a hemicellulase, or a composition comprising a cellulase or ahemicellulase. An enzyme blend/composition of the invention can comprisea mixture of two or more, three or more, or four or more enzymesselected from a xylanase, a xylosidase, a cellobiohydrolase, anarabinofuranosidase, and at least one other enzyme capable of convertinghemicellulose to monomer sugars. A non-limiting example of an enzymeblend/composition of the invention comprises a mixture of a xylanase, axylosidase, a cellobiohydrolase, an arabinofuranosidase, and aβ-glucosidase. The enzyme blend/composition of the invention is suitablyone that is non-naturally occurring.

As used herein, the term “naturally occurring composition” refers to acomposition produced by a naturally occurring source, which comprisesone or more enzymatic components or activities, wherein each of thesecomponents or activities is found at the ratio and level produced by thenaturally-occuring source as it is found in nature, untouched andunmodified by the human hand. Accordingly, a naturally occurringcomposition is, for example, one that is produced by an organismunmodified with respect to the cellulolytic or hemicelluloytic enzymessuch that the ratio or levels of the component enzymes are unalteredfrom that produced by the native organism in its native environment. A“non-naturally occurring composition,” on the other hand, refers to acomposition produced by: (1) combining component cellulolytic orhemicelluloytic enzymes either in a naturally occurring ratio or anon-naturally occurring, i.e., altered, ratio; or (2) modifying anorganism to express, overexpress or underexpress one or more endogeneousor exogenous enzymes; or (3) modifying an organism such that at leastone endogenous enzyme is deleted. A “non-naturally occurringcomposition” can also refer to a composition produced by anaturally-occurring and unmodified organism, but cultured in a man-mademedium or environment that is different from the organism's nativeenvironment, such that the amounts or weight ratios of particularenzymes in the composition differ from those existing in a compositionmade by a native organism grown in its native habitat.

The enzyme blend/composition of the invention described herein is, forexample, a fermentation broth. The fermentation broth can be one of afilamentous fungus, including, e.g., a Trichoderma, Humicola, Fusarium,Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. Anexemplary fungus of Trichoderma spp. is a Trichoderma reesei. Anexemplary fungus of Penicillium spp. is a Penicillium funiculosum. Thefermentation broth can be, for example, a cell-free fermentation brothor a whole cell broth.

The enzyme blend/composition of the invention described herein is, inanother example, a cellulase compostion. The cellulase composition is afilamentous fungal cellulase composition, including, for example, aTrichoderma, such as a Trichoderma reesei, cellulase composition. Thecellulase composition can be produced by a filamentous fungus, forexample, a Trichoderma, such as a Trichoderma reesei.

For example, an enzyme blend/composition of the invention can comprise(a) one or more xylanase enzyme(s), wherein at least one of said one ormore xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichodermareesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or more β-xylosidaseenzyme(s), wherein at least one of said one or more β-xylosidaseenzyme(s) is a Group 1 β-xylosidase or a Group 2 β-xylosidase, whereinthe Group 1 β-xylosidase is an Fv3A or an Fv43A, and the Group 2β-xylosidase is a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, anFv43B, a Pa51A, a Gz43A, or a Trichoderma reesei Bxl1; (c) one or moreL-α-arabinofuranosidase enzyme(s), wherein at least one of said one ormore L-α-arabinofuranosidase is an Af43A, an Fv43B, a Pf51A, a Pa51A, oran Fv51A; (d) one or more cellulase enzymes; and optionally (e) one ormore other components. The enzyme blend/composition is suitably one thatis non-naturally occurring. The one or more cellulase enzyme(s) of (d)is desirably able to achieve at least 0.00005 fraction product per mgprotein per gram of phosphoric acid swollen cellulose (PASO) asdetermined by a calcofluor assay. In a non-limiting example, thecombined weight of xylanase enzyme(s) in the composition can representor constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to15 wt. %, 10 wt. % to 15 wt. %) of the combined or total protein weightin the composition, whereas the combined weight of β-xylosidaseenzyme(s) can represent or constitute 2 wt. % to 50 wt. % (e.g., 2 wt. %to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the totalproteins in the composition, whereas the combined weight ofL-α-arabinofuranosidase enzyme(s) can represent or constitute 2 wt. % to50 wt. % (e.g., 2 wt. % to 30 wt. %, 2 wt. % to 20 wt. %, 5 wt. % to 15wt. %, 5 wt. % to 10 wt. %) of the combined or total protein weight inthe composition, whereas the the combined weight of cellulase enzyme(s)can represent or constitute 30 wt. % to 80 wt. % (e.g., 40 wt. % to 70wt. %, 50 wt. % to 60 wt. %) of the combined or total protein weight inthe composition. The enzyme blend/composition as described herein is,for example, a fermentation broth composition. The fermentation brothis, for example, one of a filamentous fungus, including, withoutlimitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor,Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus ofTrichoderma spp. is a Trichoderma reesei. An exemplary fungus ofPenicillium spp. is a Penicillium funiculosum. The fermentation can be,for example, a cell-free fermentation broth or a whole cell broth. Theenzyme blend/composition as described herein can also be a cellulasecomposition, for example, a filamentous fungal cellulase composition.The cellulase composition, for example, can be produced by a filamentousfungus, such as by a Trichoderma.

For example, an enzyme blend/composition of the invention can comprise(a) one or more xylanase enzyme(s) wherein at least one of said one ormore xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichodermareesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both of Group 1β-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2β-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, anFv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reeseiBxl1; (d) one or more cellulase enzyme(s); and optionally (e) one ormore other components. The one or more celluase enzyme(s) of (e) isdesirably able to to achieve at least 0.00005 fraction product per mgprotein per gram of phosphoric acid swollen cellulose (PASO) asdetermined by a calcofluor assay. The enzyme blend/composition issuitably one that is non-naturally occurring. In a non-limiting example,the combined weight of xylanase enzyme(s) in the composition canrepresent or constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %,5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the combined or totalprotein weight in the composition, whereas the combined weight of theGroup 1 β-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g.,2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of thetotal protein weight in the composition, whereas the combined weight ofthe Group 2 β-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. %(e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) ofthe total protein weight in the composition, and wherein the combinedweight of the cellulase enzyme(s) can represent or constitute 30 wt. %to 80 wt. % (e.g., 40 wt. % to 70 wt. %, 50 wt. % to 60 wt. %) of thecombined or total protein weight in the composition. The ratio of theweight of Group 1 β-xylosidase enzymes to the weight of Group 2β-xylosidase enzymes can be, for example, 1:10 to 10:1 (e.g., 1:8 to8:1, 1:6 to 6:1, 1:4 to 4:1, 1:2 to 2:1, or 1:1). The enzymeblend/composition can further comprise additional components, which maybe accessory proteins or other protein/non-protein components. Theadditional components can constitute, for example, 1 wt. % to 50 wt. %,1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. %to 20 wt. % of the total weight of proteins in the composition. Theenzyme blend/composition as described herein is, for example, afermentation broth composition. The fermentation broth is, for example,one of a filamentous fungus, including, without limitation, aTrichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus,Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp.is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is aPenicillium funiculosum. The fermentation can be, for example, acell-free fermentation broth or a whole cell broth. The enzymeblend/composition as described herein can also be a cellulasecomposition, for example, a filamentous fungal cellulase composition.The cellulase composition, for example, can be produced by a filamentousfungus, such as by a Trichoderma.

In further examples, an enzyme blend/composition of the invention cancomprise (a) one or more xylanase enzyme(s) wherein at least one of saidone or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, aTrichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or moreβ-xylosidase enzyme(s) wherein at least one of the one or moreβ-xylosidase enzyme(s) is a Group 1 β-xylosidase enzyme Fv3A or Fv43A ora Group 2 β-xylosidase enzyme Pf43A, an Fv43D, an Fv39A, an Fv43E, anFo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; (c)one or more L-α-arabinofuranosidase enzyme(s), wherein at least one ofsaid one or more L-α-arabinofuranosidase enzyme(s) is an Af43A, anFv43B, a Pf51A, or an Fv51A; (d) one or more β-glucosidase enzyme(s);and optionally (e) one or more other components. The enzymeblend/composition is suitably one that is non-naturally occurring. In anon-limiting example, the combined weight of xylanase enzyme(s) in thecomposition can represent or constitute 5 wt. % to 45 wt. % (e.g., 5 wt.% to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of thecombined or total protein weight in the composition, whereas thecombined weight of the β-xylosidase enzyme(s) can constitute 2 wt. % to50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10wt. %) of the total protein weight in the composition, whereas thecombined weight of the L-α-arabinofuranosidase enzyme(s) can constitute2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5wt. % to 10 wt. %) of the total protein weight in the composition, andwherein the combined weight of the β-glucosidase enzyme(s) canconstitute 2 wt. % to 50 wt. % (e.g., up to 50 wt. %, 2 wt. % to 10 wt.%, or 3 wt. % to 8 wt. %) of the combined or total protein weight in thecomposition. The enzyme blend/composition can further compriseadditional components, which may be accessory proteins or otherprotein/non-protein components. The additional components canconstitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt.% to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the totalweight of proteins in the composition. The enzyme blend/composition asdescribed herein is, for example, a fermentation broth composition. Thefermentation broth is, for example, one of a filamentous fungus,including, without limitation, a Trichoderma, Humicola, Fusarium,Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. Anexemplary fungus of Trichoderma spp. is a Trichoderma reesei. Anexemplary fungus of Penicillium spp. is a Penicillium funiculosum. Thefermentation can be, for example, a cell-free fermentation broth or awhole cell broth. The enzyme blend/composition as described herein canalso be a cellulase composition, for example, a filamentous fungalcellulase composition. The cellulase composition, for example, can beproduced by a filamentous fungus, such as by a Trichoderma.

An enzyme blend/composition of the invention can also comprise, forexample, (a) one or more xylanase enzyme(s) wherein at least one of saidone or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, aTrichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both ofGroup 1 β-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2β-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, anFv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reeseiBxl1; and (d) one or more β-glucosidase enzyme(s); and optionally (e)one or more other components. The enzyme blend/composition is suitablyone that is non-naturally occurring. In a non-limiting example, thecombined weight of xylanase enzyme(s) constitutes 5 wt. % to 45 wt. %(e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %)of the total weight of proteins in the composition, whereas the combinedweight of Group 1 β-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. %(e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) ofthe total weight of proteins in the composition, whereas the combinedweight of Group 2 β-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. %(e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) ofthe total weight of proteins in the composition, whereas the combinedweight of β-glucosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g.,2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of thetotal weight of proteins in the composition. The ratio of the weight ofGroup 1 β-xylosidase enzymes to the weight of Group 2 β-xylosidaseenzymes can be, for example, 1:10 to 10:1, for example, 1:8 to 8:1, 1:6to 6:1, 1:4 to 4:1, 1:2 to 2:1, or 1:1. The enzyme blend/composition canfurther comprise additional components, which may be accessory proteinsor other protein/non-protein components. The additional components canconstitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt.% to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the totalweight of proteins in the composition. The enzyme blend/composition asdescribed herein is, for example, a fermentation broth composition. Thefermentation broth is, for example, one of a filamentous fungus,including, without limitation, a Trichoderma, Humicola, Fusarium,Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. Anexemplary fungus of Trichoderma spp. is a Trichoderma reesei. Anexemplary fungus of Penicillium spp. is a Penicillium funiculosum. Thefermentation can be, for example, a cell-free fermentation broth or awhole cell broth. The enzyme blend/composition as described herein canalso be a cellulase composition, for example, a filamentous fungalcellulase composition. The cellulase composition, for example, can beproduced by a filamentous fungus, such as by a Trichoderma.

Moreover, an enzyme blend/composition of the invention can comprise, forexample, (a) one or more xylanase enzyme(s) wherein at least one of saidone or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, aTrichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or moreβ-xylosidase enzyme(s) wherein at least one of said one or moreβ-xylosidase enzyme(s) is a Group 1 β-xylosidase or a Group 2β-xylosidase, wherein Group 1 β-xylosidase can be an Fv3A or an Fv43A,and a Group 2 β-xylosidase can be a Pf43A, an Fv43D, an Fv39A, an Fv43E,an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1;and (c) one or more L-α-arabinofuranosidase enzyme(s), wherein at leastone of said one or more L-α-arabinofuranosidase enzyme(s) is an Af43A,an Fv43B, a Pf51A, or an Fv51A; and optionally (d) one or more othercomponents. The enzyme blend/composition is suitably one that isnon-naturally occurring. In a non-limiting example, the combined weightof the xylanase enzyme(s) constitutes 5 wt. % to 45 wt. % (e.g., 5 wt. %to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the totalprotein weight in the composition, whereas the combined weight of theβ-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total proteinweight of the composition, whereas the combined weight ofL-α-arabinofuranosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g.,2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of thetotal protein weight of the composition. The enzyme blend/compositioncan further comprise additional components, which may be accessoryproteins or other protein/non-protein components. The additionalcomponents can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt.% of the total weight of proteins in the composition. The enzymeblend/composition as described herein is, for example, a fermentationbroth composition. The fermentation broth is, for example, one of afilamentous fungus, including, without limitation, a Trichoderma,Humicola, Fusarium, Aspergillus, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus,Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp.is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is aPenicillium funiculosum. The fermentation can be, for example, acell-free fermentation broth or a whole cell broth. The enzymeblend/composition as described herein can also be a cellulasecomposition, for example, a filamentous fungal cellulase composition.The cellulase composition, for example, can be produced by a filamentousfungus, such as by a Trichoderma.

An enzyme blend/composition of the invention can also be one thatcomprises (a) one or more xylanase enzyme(s) wherein at least one ofsaid one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, aTrichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both ofGroup 1 β-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2β-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, anFv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reeseiBxl1; and optionally (d) one or more other components. The enzymeblend/composition is suitably one that is non-naturally occurring. In anon-limiting example, the combined weight of xylanase enzyme(s) canconstitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15wt. %, 10 wt. % to 15 wt. %) of the total protein weight in thecomposition, whereas the combined weight of Group 1 β-xylosidaseenzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %,5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight inthe composition, wheras the combined weight of Group 2 β-xylosidaseenzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %,5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight inthe composition. The ratio of the weight of Group 1 β-xylosidase enzymesto the weight of Group 2 β-xylosidase enzymes can be, for example, 1:10to 10:1, for example, 1:8 to 8:1, 1:6 to 6:1, 1:4 to 4:1, 1:2 to 2:1, or1:1. The enzyme blend/composition can further comprise additionalcomponents, which may be accessory proteins or other protein/non-proteincomponents. The additional components can constitute, for example, 1 wt.% to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in thecomposition. The enzyme blend/composition as described herein is, forexample, a fermentation broth composition. The fermentation broth is,for example, one of a filamentous fungus, including, without limitation,a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus,Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp.is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is aPenicillium funiculosum. The fermentation can be, for example, acell-free fermentation broth or a whole cell broth. The enzymeblend/composition as described herein can also be a cellulasecomposition, for example, a filamentous fungal cellulase composition.The cellulase composition, for example, can be produced by a filamentousfungus, such as by a Trichoderma.

The enzymes, enzyme blends/compositions of the disclosure can be used inthe food industry, e.g., for baking, for fruit and vegetable processing,in breaking down of agricultural waste, in the manufacture of animalfeed, in pulp and paper production, in textile manufacture, or inhousehold and industrial cleaning agents. The enzymes, and the enzymesin the enzyme blends/compositions of the disclosure are, for example,each independently produced by a microorganism, e.g., by a fungi or abacteria.

The enzymes, enzyme blends/compositions of the disclosure can also beused as commercial enzymes or compositions to digest lignocellulose fromany suitable sources, including all biological sources, such as plantbiomasses, e.g., corn, grains, grasses (e.g., Indian grass, such asSorghastrum nutans; or, switchgrass, e.g., Panicum species, such asPanicum virgatum), or, woods or wood processing byproducts, e.g., in thewood processing, pulp and/or paper industry, in textile manufacture, inhousehold and industrial cleaning agents, and/or in biomass wasteprocessing.

In another aspect, the disclosure provides isolated, synthetic orrecombinant nucleic acids having at least about 70%, for example, atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or complete (100%) sequence identity to a nucleic acidsequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of atleast about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,or 2000, residues. Relatedly, the disclosure provides isolated,synthetic, or recombinant nucleic acids that are capable of hybridizing,under high stringency conditions, to a complement of 97%, 98%, 99%, orcomplete (100%) sequence identity to a nucleic acid sequence of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 46, 47, 48, 49, or 50, or to a fragment thereof. Thefragment, for example, can be at least 150 contiguous residues inlength, for example, at least 200, 250, or 300 contiguous residues inlength. The present disclosure provides nucleic acids encoding apolypeptide having hemicellulolytic activity. Exemplary hemicellolyticactivity includes, without limitation, xylanase, β-xylosidase, and/orL-α-arabinofuranosidase activity. Exemplary polypeptides havinghemicellulolytic activity include, without limitation, a xylanase, aβ-xylosidase, and/or an L-α-arabinofuranosidase.

The disclosure further provides isolated, synthetic, or recombinantnucleic acids encoding an enzyme of the disclosure, including apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43,or 44, or a subsequence thereof (e.g., a catalytic domain (“CD”) orcarbohydrate binding module (“CBM”)), or a suitable variant thereof. Insome embodiments, a nucleic acid of the disclosure encodes the matureportion of a protein of amino acid sequence SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or44, which is optionally operably linked to a heterologous signalsequence, e.g., the Trichoderma reesei CBHI signal sequence. The nucleicacid desirably encodes a polypeptide having hemicellulolytic activity,e.g., xylanase, β-xylosidase, and/or L-α- arabinofuranosidase activity.The nucleic acid of the disclosure encodes a hemicellulase, for example,a xylanase, a β-xylosidase, and/or an L-α-arabinofuranosidase, or asuitable variant thereof. Further nucleic acids of the disclosure aredescribed in Section 6.2 below.

The disclosure additionally provides expression cassettes comprising anucleic acid of the disclosure or a subsequence thereof. For example,the nucleic acid comprises at least about 70%, e.g., at least about 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49,or 50, over a region of at least about 10 residues, e.g., at least about10, 20, 30, 40, 50, 75, 90, 100, 150, 200, 250, 300, 350, 400, or 500residues. In another example, the nucleic acid encodes a polypeptide ofSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 43, or 44, wherein the nucleic acid is optionallyoperably linked to a promoter. The promoter can be, e.g., a fungal,viral, bacterial, mammalian, or plant promoter. The promoter can be aconstitutive promoter or an inducible promoter. An exemplary suitablepromoter is expressable in filamentous fungi, e.g., Trichoderma reesei.A suitable promoter can be derived from a filamentous fungus, e.g.,Trichoderma reesei, e.g., a cellobiohydrolase I (“cbh1”) gene promoterfrom Trichoderma reesei.

The disclosure further provides a recombinant cell engineered to expressa nucleic acid of the disclosure or an expression cassette of thedisclosure. For example, the nucleic acid comprises at least about 70%,e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to a nucleic acid sequence of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10,e.g., at least about 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400,or 500 nucleotide residues. The nucleic acid can encode a polypeptide ofSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 43, or 44, wherein the nucleic acid is optionallyoperably linked to a promoter. The expression cassette can comprise thenucleic acid having at least about 70% (e.g., at least about 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequenceidentity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of atleast about 10 nucleotide residues. For example, the expression cassettecan comprise the nucleic acid encoding a polypeptide of SEQ ID NO:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 43, or 44, wherein the nucleic acid is optionally operably linked toa promoter. The recombinant cell is desirably a recombinant bacterialcell, a recombinant mammalian cell, a recombinant fungal cell, arecombinant yeast cell, a recombinant insect cell, a recombinant algalcell, or a recombinant plant cell. For example, the recombinant cell isa recombinant filamentous fungal cell, such as a Trichoderma, Humicola,Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya,Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporiumcell.

The disclosure provides transgenic plants comprising a nucleic acid ofthe disclosure or an expression cassette of the disclosure.

The disclosure provides isolated, synthetic or recombinant polypeptidescomprising an amino acid sequence having at least about 80%, e.g., atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequenceidentity to a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, over a regionof at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, or 350 residues, or over the full length immaturepolypeptide, the full length mature polypeptide, the full length CD, orthe full length CBM. Exemplary polypeptides include fragments of atleast about 10, for example, at least about 15, 20, 25, 30, 35, 40, 45,50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600 residues in length. In specific embodiments, the fragmentscomprise a CD and/or a CBM. Where a fragment comprises both a CD and aCBM of an enzyme of the disclosure, the fragment optionally includes alinker separating the CD and the CBM. The linker can be a native linkeror a heterologous linker. In certain embodiments, the polypeptides ofthe disclosure have one or more hemicellulase activities. Polypeptidesor peptide sequences of the disclosure include sequences encoded by thenucleic acids of the disclosure. Exemplary polypeptides are described inSection 6.1.

The disclosure additionally provides a chimeric or fusion proteincomprising at least one domain of a polypeptide of the disclosure (e.g.,the CD, the CBM, or both). The at least one domain can be operablylinked to a second amino acid sequence, e.g., a signal peptide sequence.

Conversely, the disclosure provides a chimeric or fusion proteincomprising a signal sequence of a polypeptide of the disclosure operablylinked to a second sequence, e.g., encoding the amino acid sequence of aheterologous polypeptide that is not naturally associated with thesignal sequence. Accordingly, the disclosure provides a recombinantpolypeptide comprising residues 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, or 1 to 40 of apolypeptide of, e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44. Further exemplarychimeric or fusion polypeptides are described in Section 6.1.1.

The disclosure also provides methods of producing a recombinantpolypeptide comprising: (a) culturing a host cell engineered to expressa polypeptide of the disclosure; and (b) recovering the polypeptide. Therecovery of the polypeptide includes, e.g., recovery of the fermentationbroth comprising the polypeptide. In certain embodiments, recovery ofthe polypeptide can include further purification step(s).

The disclosure provides methods for hydrolyzing, breaking up, ordisrupting a cellooligosaccharide, an arabinoxylan oligomer, or aglucan- or cellulose-comprising composition comprising contacting thecomposition with an enzyme, enzyme blend/composition of the disclosureunder suitable conditions, wherein the enzyme, or enzymeblend/composition hydrolyzes, breaks up, or disrupts thecellooligosaccharide, arabinoxylan oligomer, or glucan- orcellulose-comprising composition.

The disclosure provides enzyme “blends” or compositions (also termed“enzyme blend/composition” herein), comprising a polypeptide of thedisclosure, or a polypeptide encoded by a nucleic acid of thedisclosure. In some embodiments, the polypeptide of the disclosure hasone or more activities selected from xylanase, β-xylosidase, andL-α-arabinofuranosidase activities. In certain embodiments, the enzymeblends/compositions are used or are useful for depolymerization ofcellulosic and hemicellulosic polymers to metabolizable carbon moieties.The enzyme blends of the disclosure can be in the form of a compositione.g., as a product of manufacture. The composition can be, e.g., aformulation, and can take the physical form of, e.g., a liquid or asolid. In exemplary embodiments, an enzyme blend/composition of thedisclosure includes a cellulase comprising at least three differentenzyme types selected from (1) an endoglucanase, (2) acellobiohydrolase, and (3) a β-glucosidase; or at least three differentenzymatic activities selected from (1) an endoglucanase activitycatalyzing the cleavage of internal β-1,4 linkages of cellulosic orhemicellulosic materials, resulting in shorter glucooligosaccharides,(2) a cellobiohydrolase activity catalyzing the cleavage and release, inan “exo” manner, of cellobiose units (e.g., β-1,4 glucose-glucosedisaccharide), and (3) a β-glucosidase activity catalyzing the releaseof glucose monomers from short cellooligosaccharides (e.g., cellobiose).Exemplary enzyme blends/compositions of the disclosure are described inSection 6.3.4. below.

In another aspect, the disclosure provides methods for processing abiomass material comprising lignocellulose comprising contacting acomposition comprising a cellulose and/or a fermentable sugar with apolypeptide of the disclosure, or a polypeptide encoded by a nucleicacid of the disclosure, or an enzyme blend/composition (e.g., a productof manufacture) of the disclosure. Suitable biomass material comprisinglignocellulose can be derived from an agricultural crop, a byproduct ofa food or feed production, a lignocellulosic waste product, a plantresidue, or a waste paper or waste paper product. The polypeptides ofthe disclosure can have one or more enzymatic activities selected fromcellulase, endoglucanase, cellobiohydrolase, β-glucosidase, xylanase,mannanase, β-xylosidase, arabinofuranosidase, and other hemicellulaseactivities. Suitable plant residue can comprise grain, seeds, stems,leaves, hulls, husks, corncobs, corn stover, straw, grasses, wood, woodchips, wood pulp and sawdust. The grasses can be, e.g., Indian grass, orswitchgrass. The grasses can also be, for example, Miscanthus. The paperwaste can be, e.g., discarded or used photocopy paper, computer printerpaper, notebook paper, notepad paper, typewriter paper, newspapers,magazines, cardboard, and various paper-based packaging materials. Thepaper waste can also be, for example, pulp.

The disclosure provides compositions (including enzymes, enzymeblends/compositions, e.g., products of manufacture of the disclosure)comprising a mixture of hemicellulose- and cellulose-hydrolyzingenzymes, and at least one biomass material. Optionally the biomassmaterial comprises a lignocellulosic material derived from anagricultural crop. Alternatively the biomass material is a byproduct ofa food or feed production. Suitable biomass material can also be alignocellulosic waste product, a plant residue, a waste paper or wastepaper product, or comprises a plant residue. The plant residue can,e.g., be one comprising grains, seeds, stems, leaves, hulls, husks,corncobs, corn stover, grasses, straw, wood, wood chips, wood pulp, orsawdust.

Exemplary grasses include, without limitation, Indian grass orswitchgrass. Exemplary grasses can also include Miscanthus. Exemplarypaper waste include, without limitation, discarded or used photocopypaper, computer printer paper, notebook paper, notepad paper, typewriterpaper, newspapers, magazines, cardboard and paper-based packagingmaterials. Exemplary paper waste can also include, e.g., pulp.

All publically available information as of the filing date, including,e.g., publications, patents, patent applications, GenBank sequences, andATCC deposits cited herein are hereby expressly incorporated byreference.

5. BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIGS. 1A-1B: FIG. 1A: Fv3A nucleotide sequence (SEQ ID NO:1). FIG. 1B:Fv3A amino acid sequence (SEQ ID NO:2). SEQ ID NO:2 is the sequence ofthe immature Fv3A. Fv3A has a predicted signal sequence corresponding topositions 1 to 23 of SEQ ID NO:2 (underlined); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to positions 24 to 766 of SEQ ID NO:2. The predictedconserved domain is in boldface type.

FIGS. 2A-2B: FIG. 2A: Pf43A nucleotide sequence (SEQ ID NO:3). FIG. 2B:Pf43A amino acid sequence (SEQ ID NO:4). SEQ ID NO:4 is the sequence ofthe immature Pf43A. Pf43A has a predicted signal sequence correspondingto positions 1 to 20 of SEQ ID NO:4 (underlined); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to positions 21 to 445 of SEQ ID NO:4. The predictedconserved domain is in boldface type, the predicted carbohydrate bindingmodule (“CBM”) is in uppercase type, and the predicted linker separatingthe CD and CBM is in italics.

FIGS. 3A-3B: FIG. 3A: Fv43E nucleotide sequence (SEQ ID NO:5). FIG. 3B:Fv43E amino acid sequence (SEQ ID NO:6). SEQ ID NO:6 is the sequence ofthe immature Fv43E. Fv43E has a predicted signal sequence correspondingto positions 1 to 18 of SEQ ID NO:6 (underlined); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to positions 19 to 530 of SEQ ID NO:6. The predictedconserved domain is in boldface type.

FIGS. 4A-4B: FIG. 4A: Fv39A nucleotide sequence (SEQ ID NO:7). FIG. 4B:Fv39A amino acid sequence (SEQ ID NO:8). SEQ ID NO:8 is the sequence ofthe immature Fv39A. Fv39A has a predicted signal sequence correspondingto positions 1 to 19 of SEQ ID NO:8 (underlined); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to positions 20 to 439 of SEQ ID NO:8. The predictedconserved domain is in boldface type.

FIGS. 5A-5B: FIG. 5A: Fv43A nucleotide sequence (SEQ ID NO:9). FIG. 5B:Fv43A amino acid sequence (SEQ ID NO:10). SEQ ID NO:10 is the sequenceof the immature Fv43A. Fv43A has a predicted signal sequencecorresponding to positions 1 to 22 of SEQ ID NO:10 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 23 to 449 of SEQ ID NO:10.The predicted conserved domain is in boldface type, the predicted CBM isin uppercase type, and the predicted linker separating the conserveddomain and CBM is in italics.

FIGS. 6A-6B: FIG. 6A: Fv43B nucleotide sequence (SEQ ID NO:11). FIG. 6B:Fv43B amino acid sequence (SEQ ID NO:12). SEQ ID NO:12 is the sequenceof the immature Fv43B. Fv43B has a predicted signal sequencecorresponding to positions 1 to 16 of SEQ ID NO:12 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 17 to 574 of SEQ ID NO:12.The predicted conserved domain is in boldface type.

FIGS. 7A-7B: FIG. 7A: Pa51A nucleotide sequence (SEQ ID NO:13). FIG. 7B:Pa51A amino acid sequence (SEQ ID NO:14). SEQ ID NO:14 is the sequenceof the immature Pa51A. Pa51A has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:14 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 676 of SEQ ID NO:14.The predicted L-α-arabinofuranosidase conserved domain is in boldfacetype. For expression purposes, the genomic DNA was codon optimized forexpression in T. reesei (see FIG. 60B).

FIGS. 8A-8B: FIG. 8A: Gz43A nucleotide sequence (SEQ ID NO:15). FIG. 8B:Gz43A amino acid sequence (SEQ ID NO:16). SEQ ID NO:16 is the sequenceof the immature Gz43A. Gz43A has a predicted signal sequencecorresponding to positions 1 to 18 of SEQ ID NO:16 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 19 to 340 of SEQ ID NO:16.The predicted conserved domain is in boldface type. For expressionpurposes, the Gz43A predicted signal sequence was replaced by the T.reesei CBH1 signal sequence (myrklavisaflatara (SEQ ID NO: 51)) in T.reesei (see FIG. 61).

FIGS. 9A-9B: FIG. 9A: Fo43A nucleotide sequence (SEQ ID NO:17). FIG. 9B:Fo43A amino acid sequence (SEQ ID NO:18). SEQ ID NO:18 is the sequenceof the immature Fo43A. Fo43A has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:18 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 348 of SEQ ID NO:18.The predicted conserved domain is in boldface type. For expressionpurposes, the Fo43A predicted signal sequence was replaced by the T.reesei CBH1 signal sequence (myrklavisaflatara (SEQ ID NO:51)) (see FIG.62).

FIGS. 10A-10B: FIG. 10A: Af43A nucleotide sequence (SEQ ID NO:19). FIG.10B: Af43A amino acid sequence (SEQ ID NO:20). SEQ ID NO:20 is thesequence of the immature Af43A. The predicted conserved domain is inboldface type.

FIGS. 11A-11B: FIG. 11A: Pf51A nucleotide sequence (SEQ ID NO:21). FIG.11B: Pf51A amino acid sequence (SEQ ID NO:22). SEQ ID NO:22 is thesequence of the immature Pf51A. Pf51A has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:22 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 642 of SEQ ID NO:22.The predicted L-α-arabinofuranosidase conserved domain is in boldfacetype. For expression purposes, the predicted Pf51A signal sequence wasreplaced by a codon optimized the T. reesei CBH1 signal sequence(myrklavisaflatara (SEQ ID NO:51)) (underlined) and the Pf51A nucleotidesequence was codon optimized for expression in T. reesei (see FIG. 63).

FIGS. 12A-12B: FIG. 12A: AfuXyn2 nucleotide sequence (SEQ ID NO:23).FIG. 12B: AfuXyn2 amino acid sequence (SEQ ID NO:24). SEQ ID NO:24 isthe sequence of the immature AfuXyn2. AfuXyn2 has a predicted signalsequence corresponding to positions 1 to 18 of SEQ ID NO:24(underlined); cleavage of the signal sequence is predicted to yield amature protein having a sequence corresponding to positions 19 to 228 ofSEQ ID NO:24. The predicted GH11 conserved domain is in boldface type.

FIGS. 13A-13B: FIG. 13A: AfuXyn5 nucleotide sequence (SEQ ID NO:25).FIG. 13B: AfuXyn5 amino acid sequence (SEQ ID NO:26). SEQ ID NO:26 isthe sequence of the immature AfuXyn5. AfuXyn5 has a predicted signalsequence corresponding to positions 1 to 19 of SEQ ID NO:26(underlined); cleavage of the signal sequence is predicted to yield amature protein having a sequence corresponding to positions 20 to 313 ofSEQ ID NO:26. The predicted GH11 conserved domain is in boldface type.

FIGS. 14A-14B: FIG. 14A: Fv43D nucleotide sequence (SEQ ID NO:27). FIG.14B: Fv43D amino acid sequence (SEQ ID NO:28). SEQ ID NO:28 is thesequence of the immature Fv43D. Fv43D has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:28 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 350 of SEQ ID NO:28.The predicted conserved domain is in boldface type.

FIGS. 15A-15B: FIG. 15A: Pf43B nucleotide sequence (SEQ ID NO:29). FIG.15B: Pf43B amino acid sequence (SEQ ID NO:30). SEQ ID NO:30 is thesequence of the immature Pf43B. Pf43B has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:30 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 321 of SEQ ID NO:30.The predicted conserved domain is in boldface type.

FIGS. 16A-16B: FIG. 16A: Fv51A nucleotide sequence (SEQ ID NO:31). FIG.16B: Fv51A amino acid sequence (SEQ ID NO:32). SEQ ID NO:32 is thesequence of the immature Fv51A. Fv51A has a predicted signal sequencecorresponding to positions 1 to 19 of SEQ ID NO:32 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 20 to 660 of SEQ ID NO:32.The predicted L-α-arabinofuranosidase conserved domain is in boldfacetype.

FIGS. 17A-17B: FIG. 17A: Cg51B nucleotide sequence (SEQ ID NO:33). FIG.17B: Cg51B amino acid sequence (SEQ ID NO:34). SEQ ID NO:34 is thesequence of the immature Cg51B. Cg51B has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:34 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 670 of SEQ ID NO:34.The predicted conserved domain is in boldface type.

FIGS. 18A-18B: FIG. 18A: Fv43C nucleotide sequence (SEQ ID NO:35). FIG.18B: Fv43C amino acid sequence (SEQ ID NO:36). SEQ ID NO:36 is thesequence of the immature Fv43C. Fv43C has a predicted signal sequencecorresponding to positions 1 to 22 of SEQ ID NO:36 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 23 to 333 of SEQ ID NO:36.The predicted conserved domain is in boldface type.

FIGS. 19A-19B: FIG. 19A: Fv30A nucleotide sequence (SEQ ID NO:37). FIG.19B: Fv30A amino acid sequence (SEQ ID NO:38). SEQ ID NO:38 is thesequence of the immature Fv30A. Fv30A has a predicted signal sequencecorresponding to positions 1 to 19 of SEQ ID NO:38 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 20 to 537 of SEQ ID NO:38.

FIGS. 20A-20B: FIG. 20A: Fv43F nucleotide sequence (SEQ ID NO:39). FIG.20B: Fv43F amino acid sequence (SEQ ID NO:40). SEQ ID NO:40 is thesequence of the immature Fv43F. Fv43F has a predicted signal sequencecorresponding to positions 1 to 20 of SEQ ID NO:40 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 21 to 315 of SEQ ID NO:40.

FIGS. 21A-21B: FIG. 21A: Trichoderma reesei Xyn3 nucleotide sequence(SEQ ID NO:41). FIG. 21B: Trichoderma reesei Xyn3 amino acid sequence(SEQ ID NO:42). SEQ ID NO:42 is the sequence of the immature Trichodermareesei Xyn3. Trichoderma reesei Xyn3 has a predicted signal sequencecorresponding to positions 1 to 16 of SEQ ID NO:42 (underlined);cleavage of the signal sequence is predicted to yield a mature proteinhaving a sequence corresponding to positions 17 to 347 of SEQ ID NO:42.The predicted conserved domain is in bold face type.

FIG. 22: Amino acid sequence of Trichoderma reesei Xyn2 (SEQ ID NO:43).The signal sequence is underlined. The predicted conserved domain is inbold face type. The coding sequence can be found in Törrönen et al.Biotechnology, 1992, 10:1461-65.

FIG. 23: Amino acid sequence of Trichoderma reesei Bxl1 (SEQ ID NO:44).The signal sequence is underlined. The predicted conserved domain is inbold face type. The coding sequence can be found in Margolles-Clark etal. Appl. Environ. Microbiol. 1996, 62(10):3840-46.

FIG. 24: Amino acid sequence of Trichoderma reesei Bgl1 (SEQ ID NO:45).The signal sequence is underlined. The predicted conserved domain is inbold face type. The coding sequence can be found in Barnett et al.Bio-Technology, 1991, 9(6):562-567.

FIG. 25: Cellulase activity assay using PASO hydrolysis with calcofluordetection.

FIG. 26: Xylanase elution profile.

FIG. 27: SDS-PAGE detection of the two step separation of AfuXyn 5. Lane1: Crude sample; Lane 2: Eluate from Phenyl column; Lane 3: Eluate fromGF column.

FIG. 28: pENTR/D-TOPO plasmid.

FIG. 29: pTrex3gM.

FIGS. 30A-30B: Performance of different enzymes on corncob substrate.Error bars represent the experimental errors associated with triplicatecob assays. The numbers in parenthesis along the x-axis represent theenzyme doses in mg of protein per g of cellulose.

FIG. 31: Performance of different enzyme blends/compositions on corncobsubstrate. Error bars represent the experimental errors associated withtriplicate cob assays. The numbers in parentheses along the x-axisrepresent the enzyme doses in mg of protein per g cellulose.

FIG. 32: Performance of different enzyme blends/compositions on corncobsubstrate. Error bars represent the experimental errors associated withtriplicate cob assays. The numbers in parentheses along the x-axisrepresent the enzyme doses in mg of protein per g cellulose.

FIG. 33: Performance of different enzyme blends/compositions on corncobsubstrate. Error bars represent the experimental errors associated withtriplicate cob assays. The numbers in parentheses along the x-axisrepresent the enzyme doses in mg of protein per g cellulose.

FIG. 34: Performance of different enzyme blends/compositions on corncobsubstrate. Error bars represent the experimental errors associated withtriplicate cob assays. The numbers in parentheses along the x-axisrepresent the enzyme doses in mg of protein per g cellulose.

FIGS. 35A-35C: Performance of different enzyme blends/compositions oncorncob substrate. Error bars represent the experimental errorsassociated with triplicate cob assays. The numbers along the x-axisrepresent the enzyme doses in mg of protein per g cellulose.

FIG. 36: Anomeric proton NMR region of short arabinoxylan oligomersshows cleavage by Fv43A plus Fv43B.

FIG. 37: Anomeric proton NMR region of short arabinoxylan oligomersshows cleavage of β-1,2-linked xylose from arabinose by Fv3A.

FIG. 38: Alignment between the amino acid sequences of Trichodermareesei β-xylosidase and Fv3A.

FIG. 39: pENTR-TOPO-Bgl1 (943/942) plasmid.

FIG. 40: pTrex3g 943/942 Bgl1 expression vector.

FIG. 41: pENTR-Trichoderma reesei Xyn3 plasmid.

FIG. 42: pTrex3g/Trichoderma reesei Xyn3 expression vector.

FIG. 43: pENTR-Fv3A plasmid.

FIG. 44: pTrex6g/Fv3A expression vector.

FIG. 45: TOPO Blunt/Pegl1-Fv43D plasmid.

FIG. 46: TOPO Blunt/Pegl1-Fv51A plasmid.

FIGS. 47A-47D: Glucan (FIG. 47A) and xylan (FIG. 47B) conversions tomonomer sugars by secreted enzyme fermentation broths from T. reeseiintegrated expression strains. The 3-day sample was analyzed for theextent of conversion of glucan and xylan to both monomer and solubleoligomer products (FIG. 47C). FIG. 47D shows a chromatographiccomparison of enzyme product from three T. reesei integrated expressionstrains. The experimental conditions are described in Example 1. Proteinratios differ across transformants and can be quantified as a percentageof the total integrated peak area. The “EGLs” marks the summed area ofendoglucanase peaks. EndoH was added to the protein sample in smallamounts as a reagent for HPLC analysis.

FIGS. 48A-48B: Saccharification increased in xylose monomer yield inresponse to hemicellulase addition to the enzyme composition produced byan integrated strain at 7 mg total protein per gram glucan+xylan inammonia pretreated cob. FIG. 48A: Constituent Fusarium verticillioideshemicellulases in the enzyme composition produced by the integratedstrain. FIG. 48B: Hemicellulases from other fungi.

FIGS. 49A-49B: Saccharification increased in glucose monomer yield inresponse to hemicellulase addition to the enzyme composition produced byan integrated strain at 7 mg total protein per gram glucan+xylan inammonia pretreated cob. FIG. 49A: Constituent Fusarium verticillioideshemicellulases in the enzyme composition produced by the integratedstrain. FIG. 49B: Hemicellulases from other fungi.

FIGS. 50A-50B: Saccharification increased in arabinose monomer yield inresponse to hemicellulase addition to the enzyme composition produced byan integrated strain at 7 mg total protein per gram glucan+xylan inammonia pretreated cob. FIG. 50A: Constituent Fusarium verticillioideshemicellulases in the enzyme composition produced by an integratedstrain. FIG. 50B: Hemicellulases from other fungi.

FIG. 51: A graphical presentation of the saccharification performanceacross pretreatment conditions. The X-axis corresponds to theexperimental results listed in Table 12. Yields are calculated based onthe theoretical amounts of glucan or xylan available in the rawswitchgrass. All yields are based on monomeric sugars released after 3days of saccharification with the enzyme cocktail.

FIG. 52: Amino acid sequence alignment of a number of GH39β-xylosidases. Underlined residues in bold face are the predictedcatalytic general acid-base residue (marked with “A” above thealignment) and catalytic nucleophile residue (marked with “N” above thealignment). Underlined residues in normal face in the bottom twosequences are within 4 angstroms of the substrate in the active sites ofthe respective 3D structures (pdb: 1uhv and 2bs9, respectively).Underlined residues in the Fv39A sequence are predicted to be within 4angstroms of a bound substrate in the active site.

FIGS. 53A-53C: Amino acid sequence alignment of a number of GH43 familyhydrolases. Amino acid residues highly conserved among members of thefamily are shown underlined and in bold face.

FIG. 54: Amino acid sequence alignment of a number of GH51 familyenzymes. Amino acid residues highly conserved among members of thefamily are shown underlined and in bold type.

FIGS. 55A-55B: Amino acid sequence alignments of a number of GH10 andGH11 family endoxylanases. FIG. 55A; Alignment of GH10 family xylanases.Underlined residues in bold face are the the catalytic nucleophileresidues (marked with “N” above the alignment). FIG. 55B; Alignment ofGH11 family xylanases. Underlined residues in bold face are the thecatalytic nucleophile residues and general acid base residues (markedwith “N” and “A”, respectively, above the alignment).

FIGS. 56A-56B: Saccharification of dilute ammonia pretreated switchgrasswith various enzyme blends/compositions; FIG. 56A: glucan conversion;FIG. 56B: xylan conversion. The numbers below the figures on the x-axisrefer to the amount of total mg of each protein in a givenblend/composition per g of glucan or xylan, as described in Example 13.

FIGS. 57A-57B: Saccharification of dilute ammonia pretreated switchgrasswith an enzyme composition produced by integrated strain H3A; FIG. 57A:glucan conversion; FIG. 57B: xylan conversion. Experimental conditionsare described in Example 14.

FIGS. 58A-58C: Saccharification of steam-expanded sugarcane bagasse withan enzyme composition produced by integrated strain H3A at differentenzyme doses; FIG. 58A: glucan conversion; FIG. 58B: xylan conversion;FIG. 58C: 3-day glucan and xylan conversions. Experimental conditionsare described in Example 17.

FIGS. 59A-59C: Saccharification of dilute-acid pretreated corn fiberwith various enzymes or enzyme blends; FIG. 59A: glucan conversion; FIG.59B: xylan conversion: FIG. 59C: 5-day glucan and xylan conversions. Theadjusted sugar (glucose or xylose) reflected the sugar being producedfrom the enzymatic step minus the starting sugar levels. Ratios shownalong the x-axis represent the enzyme dose in mg of total protein pergram of cellulose. Experimental conditions are described in Example 18.

FIGS. 60A-60B: FIG. 60A: Deduced cDNA for Pa51A (SEQ ID NO:46). FIG.60B: Codon optimized cDNA for Pa51A (SEQ ID NO:47).

FIG. 61: Coding sequence for a construct comprising a CBH1 signalsequence (underlined) upstream of genomic DNA encoding mature Gz43A (SEQID NO:48).

FIG. 62: Coding sequence for a construct comprising a CBH1 signalsequence (underlined) upstream of genomic DNA encoding mature Fo43A (SEQID NO:49).

FIG. 63: Codon optimized coding sequence for a construct comprising aCBH1 signal sequence (underlined) upstream of codon optimized DNAencoding mature Pf51A (SEQ ID NO:50).

FIGS. 64A-64B: Amino acid sequence alignment of a number of GH3 familyhydrolases. Amino acid residues highly conserved among members of thefamily are shown underlined and in bold face type. The box marks thepredicted catalytic residue wtih flanking residues predicted to beinvolved in substrate binding.

FIG. 65: Amino acid sequence alignment of two representative FusariumGH30 family hydrolases. Amino acid residues that are conserved amongmembers of the family are shown underlined and in bold face type.

FIG. 66 and FIG. 67: Provide a summary of the sequence identifiers usedin the present disclosure for glycosyl hydrolase enzymes;

FIG. 68: Provides accession numbers for additional glycosyl hydrolaseenzymes referred to in the Examples.

FIG. 69: Activity of expressed proteins on synthetic substrates pNPA andpNPX (as defined in Section 7.1.6. below) in terms relative to theTrichoderma reesei Quad delete host background activity. The Quad deletehost background (or “XQuad”) is defined as the activity of the expressedprotein(s) in the Quad delete strain divided by the activity of the Quaddelete background strain without the expressed protein(s). For example,a value of >1 indicates that the expressed protein have an activitygreater than that of the background.

FIG. 70: Xylanase activity of purified candidate endo-xylanases withbirchwood xylan, incubated at 50° C., pH 5.

FIG. 71: Percent conversion of cob arabinoxylan oligomers to monomerproducts based on total sugar available as determined by acidhydrolysis. See, Example 4.

FIG. 72A and FIG. 72B: Experiment results (as described in Example 7)defining the level of hemicellulase activity for hydrolyzing treatedcorncob to monomer sugars. The column entitled “run #” indicates therandomized experimental order. The column entitled “trial #” indicatesthe standard experimental design order. The column entitled “Quad”indicates fraction of the total protein that is from the culturesupernatant for a growth of the Quad deleted T. reesei strain. Thecolumn entitled “Xyn3” indicates fraction of the total protein that isTrichoderma reesei Xyn3. The column entitled “Fv43D” indicates fractionof the total protein that is Fv43D. The column entitled “Fv51A”indicates fraction of the total protein that is Fv51A. The columnentitled “Fv43A” indicates fraction of the total protein that is Fv43A.The column entitled “Fv43B” indicates fraction of the total protein thatis Fv43B. The column entitled “loading (ug/mg carbo)” indicates theprotein loaded into the saccharification reaction in units of microgramsof protein per milligram of carbohydrate. The columns entitled “Xylmg/mL, Glu mg/mL, Arab mg/mL, and G+X+A mg/mL” indicate theconcentration of xylose, glucose, arabinose, and the combination ofthose three sugar products that is detected at the end of thesaccharification reaction. The columns entitled “Xyl % theor, Glu %theor, and Arab % theor” indicate the percent of theoretical yield ofxylose, glucose, and arabinose reached at the end of thesaccharification reaction.

FIG. 73: Calculated ratios of the seven enzymatic components forpredicted maximal yield of glucose, xylose and arabinose from hydrolysisof corncob. The column “loading total mg/gr carb” indicates the totalenzyme dose at which the predictions are calculated. The rows entitled“Total mg/ml G+X+A, % Yield Glucose, % Yield Xylose, and % YieldArabinose” indicate the response for which the optimum has beencalculated. The column entitled “r2 data fit to model (includes bothloadings)” indicates the r-squared statistical parameter for the modelfit to data presented in Table 5. The columns entitled “fractionAccellerase, fraction Quad del sup, fraction purified Trichoderma reeseiXyn3, fraction purified Fv43D, fraction purified Fv51A, fractionpurified Fv43A, and fraction purified Fv43B” indicate the fraction ofthat component that is calculated to be optimal by the model fitted tothe data in Table 5.

FIG. 74: Refinement of enzyme loadings for maximal hydrolytic conversionof corncob using blends including Fv3A and Fv43D enzymes in 1.06 g, 14%dry solids pretreated cob reactions. The conditions for saccharificationwere as described in Example 7. Columns marked with enzyme namesindicate the mg of each of the listed enzyme per gram of glucan or xylanused.

FIG. 75: Refinement of enzyme loadings for maximal conversion of corncobusing blends containing Fv51A as the only L-α-arabinofuranosidase in1.06 gr, 14% dry solids pretreated cob reactions. The conditions usedfor saccharification were as described in Example 7. Columns marked withenzyme names indicate the mg of each of the listed enzymes per gram ofglucan or xylan used. Columns marked with carbohydrates indicate the mgper mL of each carbohydrate product produced based on measurements madewith size exclusion chromatography. The >dp2 column includes alloligomers larger than a disaccharide.

FIG. 76: Sugar yields obtained from mixes A, B, C of purifiedhemicellulases tested in 1.06 g, 14% dry solids pretreated cobreactions. The conditions used for saccharification were as described inExample 7. Mix A: 6 mg Trichoderma reesei Xyn3, 4 mg Fv3A, 1 mg Fv51Aper gram xylan. Mix B: 6 mg Trichoderma reesei Xyn3, 1 mg Fv43D, 3 mgFv43A, 3 mg Fv43B per gram xylan. Mix C: 6 mg Trichoderma reesei Xyn3, 3mg Fv3A, 1 mg Fv43D, 1 mg Fv51A per gram xylan. Columns marked withmonomer sugars indicate the % yield of each of the listed monomer.

FIG. 77: Sugar yields from treatment of hemicellulose preparations madefrom corncob, sorghum, switchgrass and sugar cane bagasse byhemicellulase mixes A, B, C. The reactions were run at 100-μL scale in50 mM pH 5.0 Sodium Acetate buffer for 6 h at 48° C. as described inExample 8. The % yield for each monomer sugar is shown.

FIG. 78: Concentration of the majority enzymes expressed by various T.reesei integrated expression strains (designated H3A, 39A, 69A, A10A,G6A, 102, 44A, 11A, G9A) as determined by percent of the integrated HPLCarea.

FIG. 79: List of switchgrass pretreatment parameters andsaccharification results from the various pretreatments.

FIG. 80: Saccharification results of a hardwood pulp with the enzymecomposition produced by a T. reesei integrated strain, H3A, in reactionswith different amounts of solids, enzymes, and incubation time.

FIG. 81: Saccharification of a hardwood pulp with the enzyme compositionproduced by an integrated strain H3A over a temperature range and a pHrange.

Signal sequences listed below and in the figures were predicted. Thepredictions were made with the SignalP algorithm (available atwww.cbs.dtu.dk). Domain predictions were made based on one or more ofthe Pfam, SMART, or NCBI databases.

6. DETAILED DESCRIPTION

Enzymes have traditionally been classified by substrate specificity andreaction products. In the pre-genomic era, function was regarded as themost amenable (and perhaps most useful) basis for comparing enzymes andassays for various enzymatic activities have been well-developed formany years, resulting in the familiar EC classification scheme.Cellulases and other glycosyl hydrolases, which act upon glycosidicbonds between two carbohydrate moieties (or a carbohydrate andnon-carbohydrate moiety—as occurs in nitrophenol-glycoside derivatives)are, under this classification scheme, designated as EC 3.2.1.-, withthe final number indicating the exact type of bond cleaved. For example,according to this scheme an endo-acting cellulase (1,4-β-endoglucanase)is designated EC 3.2.1.4.

With the advent of widespread genome sequencing projects, sequencingdata have facilitated analyses and comparison of related genes andproteins. Additionally, a growing number of enzymes capable of acting oncarbohydrate moieties (i.e., carbohydrases) have been crystallized andtheir 3-D structures solved. Such analyses have identified discreetfamilies of enzymes with related sequence, which contain conservedthree-dimensional folds that can be predicted based on their amino acidsequence. Further, it has been shown that enzymes with the same orsimilar three-dimensional folds exhibit the same or similarstereospecificity of hydrolysis, even when catalyzing differentreactions (Henrissat et al., 1998, FEBS Lett 425(2): 352-4; Coutinho andHenrissat, 1999, in Genetics, biochemistry and ecology of cellulosedegradation. T. Kimura. Tokyo, Uni Publishers Co: 15-23.).

These findings form the basis of a sequence-based classification ofcarbohydrase modules, which is available in the form of an internetdatabase, the

Carbohydrate-Active enZYme server (CAZy), available atafmb.cnrs-mrs.fr/CAZY/index.html (Carbohydrate-active enzymes: anintegrated database approach. See Cantarel et al., 2009, Nucleic AcidsRes. 37 (Database issue):D233-38).

CAZy defines four major classes of carbohydrases distinguishable by thetype of reaction catalyzed: Glycosyl Hydrolases (GH's),Glycosyltransferases (GT's), Polysaccharide Lyases (PL's), andCarbohydrate Esterases (CE's). The enzymes of the disclosure areglycosyl hydrolases. GH's are a group of enzymes that hydrolyze theglycosidic bond between two or more carbohydrates, or between acarbohydrate and a non-carbohydrate moiety. A classification system forglycosyl hydrolases, grouped by sequence similarity, has led to thedefinition of over 85 different families. This classification isavailable on the CAZy web site.

The enzymes of the disclosure belong, inter alia, to the glycosylhydrolase families 3, 10, 11, 30, 39, 43, and/or 51.

Glycoside hydrolase family 3 (“GH3”) enzymes include, e.g.,β-glucosidase (EC:3.2.1.21); β-xylosidase (EC:3.2.1.37); N-acetylβ-glucosaminidase (EC:3.2.1.52); glucan β-1,3-glucosidase (EC:3.2.1.58);cellodextrinase (EC:3.2.1.74); exo-1,3-1,4-glucanase (EC:3.2.1); andβ-galactosidase (EC 3.2.1.23). For example, GH3 enzymes can be thosethat have β-glucosidase, β-xylosidase, N-acetyl β-glucosaminidase,glucan β-1,3-glucosidase, cellodextrinase, exo-1,3-1,4-glucanase, and/orβ-galactosidase activity. Generally, GH3 enzymes are globular proteinsand can consist of two or more subdomains. A catalytic residue has beenidentified as an aspartate residue that, in β-glucosidases, located inthe N-terminal third of the peptide and sits within the amino acidfragment SDW (Li et al. 2001, Biochem. J. 355:835-840). Thecorresponding sequence in Bgl1 from T. reesei is T266D267W268 (countingfrom the methionine at the starting position), with the catalyticresidue aspartate being the D267. The hydroxyl/aspartate sequence isalso conserved in the GH3 β-xylosidases tested. For example, thecorresponding sequence in T. reesei Bxl1 is S310D311 and thecorresponding sequence in Fv3A is S290D291.

Glycoside hydrolase family 39 (“GH39”) enzymes have α-L-iduronidase(EC:3.2.1.76) or β-xylosidase (EC:3.2.1.37) activity. Thethree-dimensional structure of two GH39 β-xylosidases, fromThermoanaerobacterium saccharolyticum (Uniprot Accession No. P36906) andGeobacillus stearothermophilus (Uniprot Accession No. Q9ZFM2), have beensolved (see Yang et al. J. Mol. Biol. 2004, 335(1):155-65 and Czjzek etal., J. Mol. Biol. 2005, 353(4):838-46). The most highly conservedregions in these enzymes are located in their N-terminal sections, whichhave a classic (α/β)8 TIM barrel fold with the two key active siteglutamic acids located at the C-terminal ends of β-strands 4 (acid/base)and 7 (nucleophile). Fv39A residues E168 and E272 are predicted tofunction as catalytic acid-base and nucleophile, respectively, based ona sequence alignment of the abovementioned GH39 β-xylosidases fromThermoanaerobacterium saccharolyticum and Geobacillus stearothermophiluswith Fv39A.

Glycoside hydrolase family 43 (“GH43”) enzymes include, e.g.,L-α-arabinofuranosidase (EC 3.2.1.55); β-xylosidase (EC 3.2.1.37);endo-arabinanase (EC 3.2.1.99); and/or galactan 1,3-β-galactosidase (EC3.2.1.145). For example, GH43 enzymes can have L-α-arabinofuranosidaseactivity, β-xylosidase activity, endo-arabinanase activity, and/orgalactan 1,3-β-galactosidase activity. GH43 family enzymes display afive-bladed-β-propeller-like structure. The propeller-like structure isbased upon a five-fold repeat of blades composed of four-strandedβ-sheets. The catalytic general base, an aspartate, the catalyticgeneral acid, a glutamate, and an aspartate that modulates the pKa ofthe general base were identified through the crystal structure ofCellvibriojaponicus CjAbn43A, and confirmed by site-directed mutagenesis(see Nurizzo et al. Nat. Struct. Biol. 2002, 9(9) 665-8). The catalyticresidues are arranged in three conserved blocks spread widely throughthe amino acid sequence (Pons et al. Proteins: Structure, Function andBioinformatics, 2004, 54:424-432). Among the GH43 family enzymes testedfor useful activities in biomass hydrolysis, the predicted catalyticresidues are shown as the bold and underlined residues in the sequencesof FIGS. 53A-53C. The crystal structure of the Geobacillusstearothermophylus xylosidase (Brux et al. J. Mol. Bio., 2006,359:97-109) suggests several additional residues that may be importantfor substrate binding in this enzyme. Because the GH43 family enzymestested for biomass hydrolysis had differing substrate preferences, theseresidues are not fully conserved in the sequences aligned in FIGS.53A-53C. However among the xylosidases tested, several conservedresidues that contribute to substrate binding, either throughhydrophobic interaction or through hydrogen bonding, are conserved andare noted by single underlines in FIGS. 53A-53C.

Glycoside hydrolase family 51 (“GH51”) enzymes haveL-α-arabinofuranosidase (EC 3.2.1.55) and/or endoglucanase (EC 3.2.1.4)activity. High-resolution crystal structure of a GH51L-α-arabinofuranosidase from Geobacillus stearothermophilus T-6 showsthat the enzyme is a hexamer, with each monomer organized into twodomains: an 8-barrel (β/α)and a 12-stranded β sandwich with jelly-rolltopology (see Hovel et al. EMBO J. 2003, 22(19):4922-4932). It can beexpected that the catalytic residues will be acidic and conserved acrossenzyme sequences in the family. When the amino acid sequences of Fv51A,Pf51A, and Pa51A are aligned with GH51 enzymes of more diverse sequence,8 acidic residues remain conserved. Those are shown bold and underlinedin FIG. 54.

Glycoside hydrolase family 10 (“GH10”) enzymes also have an 8-barrel(β/α) structure. They hydrolyze in an endo fashion with a retainingmechanism that uses at least one acidic catalytic residue in a generallyacid/base catalysis process (Pell et al., J. Biol. Chem., 2004, 279(10):9597-9605). Crystal structures of the GH10 xylanases of Penicilliumsimplicissimum (Uniprot P56588) and Thermoascus aurantiacus (UniprotP23360) complexed with substrates in the active sites have been solved(see Schmidt et al. Biochem., 1999, 38:2403-2412; and Lo Leggio et al.FEBS Lett. 2001, 509: 303-308). Trichoderma reesei Xyn3 residues thatare important for substrate binding and catalysis can be derived from analignment with the sequences of abovementioned GH10 xylanases fromPenicillium simplicissimum and Thermoascus aurantiacus (FIG. 55A).Trichoderma reesei Xyn3 residue E282 is predicted to be the catalyticnucleophilic residue, whereas residues E91, N92, K95, Q97, S98, H128,W132, Q135, N175, E176, Y219, Q252, H254, W312, and/or W320 arepredicted to be involved in substrate binding and/or catalysis.

Glycoside hydrolase family 11 (“GH11”) enzymes have a β-jelly rollstructure. They hydrolyze in an endo fashion with a retaining mechanismthat uses at least one acidic catalytic residue in a generally acid/basecatalysis process. Several other residues spread throughout theirstructure may contribute to stabilizing the xylose units in thesubstrate neighboring the pair of xylose monomers that are cleaved byhydrolysis. Three GH11 family endoxylanases were tested and theirsequences are aligned in FIG. 55B. E118 (or E86 in mature T. reeseiXyn2) and E209 (or E177 in mature T. reesei Xyn2) have been identifiedas catalytic nucleophile and general/acid base residues in Trichodermareesei Xyn2, respectively (see Havukainen et al. Biochem., 1996,35:9617-24).

Glycoside hydrolase family 30 (“GH30”) enzymes are retaining enzymeshaving glucosylceramidase (EC 3.2.1.45); β-1,6-glucanase (EC 3.2.1.75);β-xylosidase (EC 3.2.1.37); β-glucosidase (3.2.1.21) activity. The firstGH30 crystal structure was the Gaucher disease-related humanβ-glucocerebrosidase solved by Grabowski, Gatt and Horowitz (Crit RevBiochem Mol Biol 1990; 25(6) 385-414). GH30 have an (α/β)₈ TIM barrelfold with the two key active site glutamic acids located at theC-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile)(Henrissat B, et al. Proc Natl Acad Sci U S A, 92(15):7090-4, 1995;Jordan et al., Applied Microbiol Biotechnol, 86:1647, 2010). Glutamate162 of Fv30A is conserved in 14 of 14 aligned GH30 proteins (13bacterial proteins and one endo-b-xylanase from the fungi Biosporaaccession no. ADG62369) and glutamate 250 of Fv30A is conserved in 10 ofthe same 14, is an aspartate in another three and non-acidic in one.There are other moderately conserved acidic residues but no others areas widely conserved.

6.1 Polypeptides of the Disclosure

The disclosure provides isolated, synthetic or recombinant polypeptidescomprising an amino acid sequence having at least about 80%, e.g., atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete (100%) sequence identityto a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45, over a region of atleast about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, or 350 residues, or over the full length of the immaturepolypeptide, the full length mature polypeptide, the full length of theconserved domain, and/or the full length CBM. The conserved domain canbe a predicted catalytic domain (“CD”). Exemplary polypeptides alsoinclude fragments of at least about 10, e.g., at least about 15, 20, 25,30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, or 600 residues in length. The fragments cancomprise a conserved domain and/or a CBM. Where a fragment comprises aconserved domain and a CBM of an enzyme, the fragment optionallyincludes a linker separating the two. The linker can be a native linkeror a heterologous linker It is contemplated that the polypeptides of thedisclosure can be encoded by a nucleic acid sequence having at leastabout 85%, about 86%, about 87%, about 88%, about 89%, or about 90%sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, or by a nucleic acid sequencecapable of hybridizing under high stringency conditions to a complementof SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, or 41, or to a fragment thereof. Exemplary nucleic acidsof the disclosure are described in Section 6.2 below.

The polypeptides of the disclosure include proteins having an amino acidsequence with at least 85%, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least50 contiguous amino acid residues of the glycosyl hydrolase sequences ofSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 43, 44, or 45. For example, a polypeptide of thedisclosure can include amino acid sequences having at least 85%, e.g.,at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to at least 10, e.g., at least 11,12, 13,14, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or350 contiguous amino acid residues of the glycosyl hydrolase sequencesof SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 43, 44, or 45. The contiguous amino acid sequencecorresponds to the conserved domain and/or the CBM and/or the signalsequence.

Any of the amino acid sequences described herein can be producedtogether or in conjunction with at least 1, e.g., at least 2, 3, 5, 10,or 20 heterologous amino acids flanking each of the C- and/or N-terminalends of the specified amino acid sequence, and or deletions of at least1, e.g., at least 2, 3, 5, 10, or 20 amino acids from the C- and/orN-terminal ends of an enzyme of the disclosure.

Other variations also are within the scope of this disclosure. Forexample, one or more amino acid residues can be modified to increase ordecrease the pl of an enzyme. The change of pl value can be achieved byremoving a glutamate residue or substituting it with another amino acidresidue.

The disclosure specifically provides an Fv3A, a Pf43A, an Fv43E, anFv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, aPf51A, an AfuXyn2, an

AfuXyn5, a Fv43D, a Pf43B, Fv43B, a Fv51A, a Trichoderma reesei Xyn3, aTrichoderma reesei Xyn2, a Trichoderma reesei Bxl1, and/or a Trichodermareesei Bgl1 polypeptide. A combination of one or more of these enzymesis suitably present in the enzyme blend/composition of the invention,for example, one that is non-naturally occurring.

Fv3A: The amino acid sequence of Fv3A (SEQ ID NO:2) is shown in FIGS.1B, 38, 64A and 64B. SEQ ID NO:2 is the sequence of the immature Fv3A.Fv3A has a predicted signal sequence corresponding to residues 1 to 23of SEQ ID NO:2 (underlined); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 24 to 766 of SEQ ID NO:2. The predicted conserved domains arein boldface type in FIG. 1B. Fv3A was shown to have β-xylosidaseactivity, for example, in an enzymatic assay usingp-nitrophenyl-β-xylopyranoside, xylobiose, mixed linear xylo-oligomers,branched arabinoxylan oligomers from hemicellulose, or dilute ammoniapretreated corncob as substrates. The predicted catalytic residue isD291, while the flanking residues, S290 and C292, are predicted to beinvolved in substrate binding (FIGS. 64A and 64B). E175 and E213 areconserved across other GH3 enzymes and are predicted to have catalyticfunctions. As used herein, “an Fv3A polypeptide” refers to a polypeptideand/or to a variant thereof comprising a sequence having at least 85%,e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to at least 50, e.g., at least75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, or 700 contiguous amino acid residues among residues 24 to 766 ofSEQ ID

NO:2. An Fv3A polypeptide preferably is unaltered as compared to nativeFv3A in residues D291, S290, C292, E175, and E213. An Fv3A polypeptideis preferably unaltered in at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% of the amino acid residues that are conserved among Fv3A,Trichoderma reesei Bxl1 and/or Trichoderma reesei Bgl1, as shown in thealignment of FIGS. 64A and 64B. An Fv3A polypeptide suitably comprisesthe entire predicted conserved domain of native Fv3A as shown in FIG.1B. An exemplary Fv3A polypeptide of the invention comprises a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the mature Fv3A sequence asshown in FIG. 1B. The Fv3A polypeptide of the invention preferably hasβ-xylosidase activity.

Accordingly an Fv3A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv)395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2. The polypeptidesuitably has β-xylosidase activity.

Pf43A: The amino acid sequence of Pf43A (SEQ ID NO:4) is shown in FIGS.2B and 53A-53C. SEQ ID NO:4 is the sequence of the immature Pf43A. Pf43Ahas a predicted signal sequence corresponding to residues 1 to 20 of SEQID NO:4 (underlined in FIG. 2B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 21 to 445 of SEQ ID NO:4. The predicted conserved domain is inboldface type, the predicted CBM is in uppercase type, and the predictedlinker separating the CD and CBM is in italics in FIG. 2B. Pf43A hasbeen shown to have β-xylosidase activity, in, for example, an enzymaticassay using p-nitophenyl-β-xylopyranoside, xylobiose, mixed linearxylo-oligomers, or ammonia pretreated corncob as substrates. Thepredicted catalytic residues include either D32 or D60, D145, and E206.The C-terminal region underlined in FIGS. 53A-53C is the predicted CBM.As used herein, “a Pf43A polypeptide” refers to a polypeptide and/or avariant thereof comprising a sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300,350, or 400 contiguous amino acid residues among residues 21 to 445 ofSEQ ID NO:4. A Pf43A polypeptide preferably is unaltered as compared tothe native Pf43A in residues D32 or D60, D145, and E206. A Pf43A ispreferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of theamino acid residues that are found conserved across a family of proteinsincluding Pf43A and 1, 2, 3, 4, 5, 6, 7, or all 8 of other amino acidsequences in the alignment of FIGS. 53A-53C. A Pf43A polypeptide of theinvention suitably comprises two or more or all of the followingdomains: (1) the predicted CBM, (2) the predicted conserved domain, and(3) the linker of Pf43A as shown in FIG. 2B. An exemplary Pf43Apolypeptide of the invention comprises a sequence having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the mature Pf43A sequence as shown in FIG. 2B. ThePf43A polypeptide of the invention preferably has β-xylosidase activity.

Accordingly a Pf43A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv)21-444, (v) 302-444, (vi) 302-445, (vii) 324-444, or (viii) 324-445 ofSEQ ID NO:4. The polypeptide suitably has β-xylosidase activity.

Fv43E: The amino acid sequence of Fv43E (SEQ ID NO:6) is shown in FIGS.3B and 53. SEQ ID NO:6 is the sequence of the immature Fv43E. Fv43E hasa predicted signal sequence corresponding to residues 1 to 18 of SEQ IDNO:6 (underlined in FIG. 3B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 19 to 530 of SEQ ID NO:6. The predicted conserved domain ismarked in boldface type in FIG. 3B. Fv43E was shown to have β-xylosidaseactivity, in, for example, enzymatic assay using4-nitophenyl-β-D-xylopyranoside, xylobiose, and mixed, linearxylo-oligomers, or ammonia pretreated corncob as substrates. Thepredicted catalytic residues include either D40 or D71, D155, and E241.As used herein, “an Fv43E polypeptide” refers to a polypeptide and/or avariant thereof comprising a sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300,350, 400, 450, or 500 contiguous amino acid residues among residues 19to 530 of SEQ ID NO:6. An Fv43E polypeptide preferably is unaltered ascompared to the native Fv43E in residues D40 or D71, D155, and E241. AnFv43E polypeptide is preferably unaltered in at least 70%, 80%, 90%,95%, 98%, or 99% of the amino acid residues that are found to beconserved among a family of enzymes including Fv43E, and 1, 2, 3, 4, 5,6, 7, or all other 8 amino acid sequences in the alignment of FIGS.53A-53C. An Fv43E polypeptide suitably comprises the entire predictedconserved domain of native Fv43E as shown in FIG. 3B. An exemplary Fv43Epolypeptide comprises a sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto mature Fv43E sequence as shown in FIG. 3B. The Fv43E polypeptide ofthe invention preferably has β-xylosidase activity.

Accoringly, an Fv43E polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:6, or to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or(iv) 29-300 of SEQ ID NO:6. The polypeptide suitably has β-xylosidaseactivity.

Fv39A: The amino acid sequence of Fv39A (SEQ ID NO:8) is shown in FIGS.4B and 52. SEQ ID NO:8 is the sequence of the immature Fv39A. Fv39A hasa predicted signal sequence corresponding to residues 1 to 19 of SEQ IDNO:8 (underlined in FIG. 4B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 20 to 439 of SEQ ID NO:8. The predicted conserved domain isshown in boldface type in FIG. 4B. Fv39A was shown to have β-xylosidaseactivity in, for example, an enzymatic assay usingp-nitophenyl-β-xylopyranoside, xylobiose or mixed, linear xylo-oligomersas substrates. Fv39A residues E168 and E272 are predicted to function ascatalytic acid-base and nucleophile, respectively, based on a sequencealignment of the above-mentioned GH39 xylosidases fromThermoanaerobacterium saccharolyticum (Uniprot Accession No. P36906) andGeobacillus stearothermophilus (Uniprot Accession No. Q9ZFM2) withFv39A. As used herein, “an Fv39A polypeptide” refers to a polypeptideand/or a variant thereof comprising a sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300,350, or 400 contiguous amino acid residues among residues 20 to 439 ofSEQ ID NO:8. An Fv39A polypeptide preferably is unaltered as compared tonative Fv39A in residues E168 and E272. An Fv39A polypeptide ispreferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of theamino acid residues that are conserved among a family or enzymesincluding Fv39A and xylosidases from Thermoanaerobacteriumsaccharolyticum and Geobacillus stearothermophilus (see above). An Fv39Apolypeptide suitably comprises the entire predicted conserved domain ofnative Fv39A as shown in FIG. 4B. An exemplary Fv39A polypeptidecomprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the matureFv39A sequence as shown in FIG. 4B. The Fv39A polypeptide of theinvention preferably has β-xylosidase activity.

Accordingly, an Fv39A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:8, or to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or(iv) 145-439 of SEQ ID NO:8. The polypeptide suitably has β-xylosidaseactivity.

Fv43A: The amino acid sequence of Fv43A (SEQ ID NO:10) is provided inFIGS. 5B and 53A-53C. SEQ ID NO:10 is the sequence of the immatureFv43A. Fv43A has a predicted signal sequence corresponding to residues 1to 22 of SEQ ID NO:10 (underlined in FIG. 5B); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to residues 23 to 449 of SEQ ID NO:10. In FIG. 5B, thepredicted conserved domain is in boldface type, the predicted CBM is inuppercase type, and the predicted linker separating the CD and CBM is initalics. Fv43A was shown to have β-xylosidase activity in, for example,an enzymatic assay using 4-nitophenyl-β-D-xylopyranoside, xylobiose,mixed, linear xylo-oligomers, branched arabinoxylan oligomers fromhemicellulose, and/or linear xylo-oligomers as substrates. The predictedcatalytic residues including either D34 or D62, D148, and E209. As usedherein, “an Fv43A polypeptide” refers to a polypeptide and/or a variantthereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or400 contiguous amino acid residues among residues 23 to 449 of SEQ IDNO:10. An Fv43A polypeptide preferably is unaltered, as compared tonative Fv43A, at residues D34 or D62, D148, and E209, An Fv43Apolypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%,or 99% of the amino acid residues that are conserved among a family ofenzymes including Fv43A and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other aminoacid sequences in the alignment of FIGS. 5B and 53A-53C. An Fv43Apolypeptide suitably comprises the entire predicted CBM of native Fv43A,and/or the entire predicted conserved domain of native Fv43A, and/or thelinker of Fv43A as shown in FIG. 5B. An exemplary Fv43A polypeptidecomprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the matureFv43A sequence as shown in FIG. 5B. The Fv45A polypeptide of theinvention preferably has β-xylosidase activity.

Accordingly an Fv43A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv)23-448, (v) 303-448, (vi) 303-449, (vii) 321-448, or (viii) 321-449 ofSEQ ID NO:10. The polypeptide suitably has β-xylosidase activity.

Fv43B: The amino acid sequence of Fv43B (SEQ ID NO:12) is shown in FIGS.6B and 53. SEQ ID NO:12 is the sequence of the immature Fv43B. Fv43B hasa predicted signal sequence corresponding to residues 1 to 16 of SEQ IDNO:12 (underlined in FIG. 6B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 17 to 574 of SEQ ID NO:12. The predicted conserved domain is inboldface type in FIG. 6B. Fv43B was shown to have both β-xylosidase andL-α-arabinofuranosidase activities, in, for example, a first enzymaticassay using 4-nitophenyl-β-D-xylopyranoside andp-nitrophenyl-α-L-arabinofuranoside as substrates. It was shown, in asecond enzymatic assay, to catalyze the release of arabinose frombranched arabino-xylooligomers and to catalyze the increased xyloserelease from oligomer mixtures in the presence of other xylosidaseenzymes. The predicted catalytic residues include either D38 or D68,D151, and E236. As used herein, “an Fv43B polypeptide” refers to apolypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, or 550 contiguous amino acidresidues among residues 17 to 574 of SEQ ID NO:12. An Fv43B polypeptidepreferably is unaltered, as compared to native Fv43B, at residues D38 orD68, D151, and E236. An Fv43B polypeptide is preferably unaltered in atleast 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues thatare conserved among a family of enzymes including Fv43B and 1, 2, 3, 4,5, 6, 7, 8, or all 9 other amino acid sequences in the alignment ofFIGS. 53A-53C. An Fv43B polypeptide suitably comprises the entirepredicted conserved domain of native Fv43B as shown in FIGS. 6B and 53.An exemplary Fv43B polypeptide comprises a sequence having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the mature Fv43B sequence as shown in FIG. 6B. TheFv43B polypeptide of the present invention preferably has β-xylosidaseactivity, L-α-arabinofuranosidase activity, or both β-xylosidase andL-α-arabinofuranosidase activities.

Accordingly, an Fv43B polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:12, or to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or(iv) 27-303 of SEQ ID NO:12. The polypeptide suitably has β-xylosidaseactivity, L-α-arabinofuranosidase activity, or both β-xylosidase andL-α-arabinofuranosidase activities.

Pa51A: The amino acid sequence of Pa51A (SEQ ID NO:14) is shown in FIGS.7B and 54. SEQ ID NO:14 is the sequence of the immature Pa51A. Pa51A hasa predicted signal sequence corresponding to residues 1 to 20 of SEQ IDNO:14 (underlined in FIG. 7B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 21 to 676 of SEQ ID NO:14. The predictedL-α-arabinofuranosidase conserved domain is in boldface type in FIG. 7B.Pa51A was shown to have both β-xylosidase activity andL-α-arabinofuranosidase activity in, for example, enzymatic assays usingartificial substrates p-nitrophenyl-β-xylopyranoside andp-nitophenyl-⊐α-L-arabinofuranoside. It was shown to catalyze therelease of arabinose from branched arabino-xylo oligomers and tocatalyze the increased xylose release from oligomer mixtures in thepresence of other xylosidase enzymes. Conserved acidic residues includeE43, D50, E257, E296, E340, E370, E485, and E493. As used herein, “aPa51A polypeptide” refers to a polypeptide and/or a variant thereofcomprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to atleast 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,550, 600, or 650 contiguous amino acid residues among residues 21 to 676of SEQ ID NO:14. A Pa51A polypeptide preferably is unaltered, ascompared to native Pa51A, at residues E43, D50, E257, E296, E340, E370,E485, and E493. A Pa51A polypeptide is preferably unaltered in at least70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that areconserved among a group of enzymes including Pa51A, Fv51A, and Pf51A, asshown in the alignment of FIG. 54. A Pa51A polypeptide suitablycomprises the predicted conserved domain of native Pa51A as shown inFIG. 7B. An exemplary Pa51A polypeptide comprises a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity to the mature Pa51A sequence as shown in FIG.7B. The Pa51A polypeptide of the invention preferably has β-xylosidaseactivity, L-α-arabinofuranosidase activity, or both β-xylosidase andL-α-arabinofuranosidase activities.

Accordingly, a Pa51A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:14, or to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or(iv) 469-676 of SEQ ID NO:14. The polypeptide suitably has β-xylosidaseactivity, L-α-arabinofuranosidase activity, or both β-xylosidase andL-α-arabinofuranosidase activities.

Gz43A: The amino acid sequence of Gz43A (SEQ ID NO:16) is shown in FIGS.8B and 53A-53C. SEQ ID NO:16 is the sequence of the immature Gz43A.Gz43A has a predicted signal sequence corresponding to residues 1 to 18of SEQ ID NO:16 (underlined in FIG. 8B); cleavage of the signal sequenceis predicted to yield a mature protein having a sequence correspondingto residues 19 to 340 of SEQ ID NO:16. The predicted conserved domain isin boldface type in FIG. 8B. Gz43A was shown to have β-xylosidaseactivity in, for example, an enzymatic assay usingp-nitophenyl-β-xylopyranoside, xylobiose or mixed, and/or linearxylo-oligomers as substrates. The predicted catalytic residues includeeither D33 or D68, D154, and E243. As used herein, “a Gz43A polypeptide”refers to a polypeptide and/or a variant thereof comprising a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100,125, 150, 175, 200, 250, or 300 contiguous amino acid residues amongresidues 19 to 340 of SEQ ID NO:16. A Gz43A polypeptide preferably isunaltered, as compared to native Gz43A, at residues D33 or D68, D154,and E243. A Gz43A polypeptide is preferably unaltered in at least 70%,80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conservedamong a group of enzymes including Gz43A and 1, 2, 3, 4, 5, 6, 7, 8 orall 9 other amino acid sequences in the alignment of FIGS. 53A-53C. AGz43A polypeptide suitably comprises the predicted conserved domain ofnative Gz43A as shown in FIG. 8B. An exemplary Gz43A polypeptidecomprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the matureGz43A sequence as shown in FIG. 8B. The Gz43A polypeptide of theinvention preferably has β-xylosidase activity.

Accordingly a Gz43A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or(iv) 53-383 of SEQ ID NO:16. The polypeptide suitably has β-xylosidaseactivity.

Fo43A: The amino acid sequence of Fo43A (SEQ ID NO:18) is shown in FIGS.9B and 53A-53C. SEQ ID NO:18 is the sequence of the immature Fo43A.Fo43A has a predicted signal sequence corresponding to residues 1 to 20of SEQ ID NO:18 (underlined in FIG. 9B); cleavage of the signal sequenceis predicted to yield a mature protein having a sequence correspondingto residues 21 to 348 of SEQ ID NO:18. The predicted conserved domain isin boldface type in FIG. 9B. Fo43A was shown to have β-xylosidaseactivity in, for example, an enzymatic assay usingp-nitophenyl-β-xylopyranoside, xylobiose and/or mixed, linearxylo-oligomers as substrates. The predicted catalytic residues includeeither D37 or D72, D159, and E251. As used herein, “an Fo43Apolypeptide” refers to a polypeptide and/or a variant thereof comprisinga sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50,75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acid residuesamong residues 18 to 344 of SEQ ID NO:18. An Fo43A polypeptidepreferably is unaltered, as compared to native Fo43A, at residues D37 orD72, D159, and E251. An Fo43A polypeptide is preferably unaltered in atleast 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues thatare conserved among a group of enzymes including Fo43A and 1, 2, 3, 4,5, 6, 7, 8 or all 9 other amino acid sequences in the alignment of FIGS.53A-53C. An Fo43A polypeptide suitably comprises the predicted conserveddomain of native Fo43A as shown in FIG. 9B. An exemplary Fo43Apolypeptide comprises a sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the mature Fo43A sequence as shown in FIG. 9B. The Fo43A polypeptideof the invention preferably has β-xylosidase activity.

Accordingly an Fo43A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or(iv) 107-348 of SEQ ID NO:18. The polypeptide suitably has β-xylosidaseactivity.

Af43A: The amino acid sequence of Af43A (SEQ ID NO:20) is shown in FIGS.10B and 53A-53C. SEQ ID NO:20 is the sequence of the immature Af43A. Thepredicted conserved domain is in boldface type in FIG. 10B. Af43A wasshown to have L-α-arabinofuranosidase activity in, for example, anenzymatic assay using p-nitophenyl-⊐α-L-arabinofuranoside as asubstrate. Af43A was shown to catalyze the release of arabinose from theset of oligomers released from hemicellulose via the action ofendoxylanase. The predicted catalytic residues include either D26 orD58, D139, and E227. As used herein, “an Af43A polypeptide” refers to apolypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,175, 200, 250, or 300 contiguous amino acid residues of SEQ ID NO:20. AnAf43A polypeptide preferably is unaltered, as compared to native Af43A,at residues D26 or D58, D139, and E227. An Af43A polypeptide ispreferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of theamino acid residues that are conserved among a group of enzymesincluding Af43A and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acidsequences in the alignment of FIGS. 53A-53C. An Af43A polypeptidesuitably comprises the predicted conserved domain of native Af43A asshown in FIG. 10B. An exemplary Af43A polypeptide comprises a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:20. The Af43Apolypeptide of the invention preferably has L-α-arabinofuranosidaseactivity.

Accordingly an Af43A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:20, or to residues (i)15-558, or (ii)15-295 of SEQ ID NO:20.The polypeptide suitably has L-α-arabinofuranosidase activity.

Pf51A: The amino acid sequence of Pf51A (SEQ ID NO:22) is shown in FIGS.11B and 54. SEQ ID NO:22 is the sequence of the immature Pf51A. Pf51Ahas a predicted signal sequence corresponding to residues 1 to 20 of SEQID NO:22 (underlined in FIG. 11B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 21 to 642 of SEQ ID NO:22. The predictedL-α-arabinofuranosidase conserved domain is in boldface type in FIG.11B. Pf51A was shown to have L-α-arabinofuranosidase activity in, forexample, an enzymatic assay using 4-nitrophenyl-⊐α-L-arabinofuranosideas a substrate. Pf51A was shown to catalyze the release of arabinosefrom the set of oligomers released from hemicellulose via the action ofendoxylanase. The predicted conserved acidic residues include E43, D50,E248, E287, E331, E360, E472, and E480. As used herein, “a Pf51Apolypeptide” refers to a polypeptide and/or a variant thereof comprisinga sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, or 600contiguous amino acid residues among residues 21 to 642 of SEQ ID NO:22.A Pf51A polypeptide preferably is unaltered, as compared to nativePf51A, at residues E43, D50, E248, E287, E331, E360, E472, and E480. APf51A polypeptide is preferably unaltered in at least 70%, 80%, 90%,95%, 98%, or 99% of the amino acid residues that are conserved amongPf51A, Pa51A, and Fv51A, as shown in in the alignment of FIG. 54. APf51A polypeptide suitably comprises the predicted conserved domain ofnative Pf51A shown in FIG. 11B. An exemplary Pf51A polypeptide comprisesa sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pf51Asequence shown in FIG. 11B. The Pf51A polypeptide of the inventionpreferably has L-α-arabinofuranosidase activity.

Accordingly a Pf51A polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:22, or to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or(iv) 461-642 of SEQ ID NO:22. The polypeptide hasL-α-arabinofuranosidase activity.

AfuXyn2: The amino acid sequence of AfuXyn2 (SEQ ID NO:24) is shown inFIGS. 12B and 55B. SEQ ID NO:24 is the sequence of the immature AfuXyn2.AfuXyn2 has a predicted signal sequence corresponding to residues 1 to18 of SEQ ID NO:24 (underlined in FIG. 12B); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to residues 19 to 228 of SEQ ID NO:24. The predicted GH11conserved domain is in boldface type in FIG. 12B. AfuXyn2 was shown tohave endoxylanase activity indirectly by observing its ability tocatalyze the increased xylose monomer production in the presence ofxylobiosidase when the enzymes act on pretreated biomass or on isolatedhemicellulose. The conserved catalytic residues include E124, E129, andE215. As used herein, “an AfuXyn2 polypeptide” refers to a polypeptideand/or a variant thereof comprising a sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to at least 50, 75, 100, 125, 150, 175, or 200contiguous amino acid residues among residues 19 to 228 of SEQ ID NO:24.An AfuXyn2 polypeptide preferably is unaltered, as compared to nativeAfuXyn2, at residues E124, E129 and E215. An AfuXyn2 polypeptide ispreferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of theamino acid residues that are conserved among AfuXyn2, AfuXyn5, andTrichoderma reesei Xyn2, as shown in the alignment of FIG. 55B. AnAfuXyn2 polypeptide suitably comprises the entire predicted conserveddomain of native AfuXyn2 shown in FIG. 12B. An exemplary AfuXyn2polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the mature AfuXyn2 sequence shown in FIG. 12B. The AfuXyn2polypeptide of the invention preferably has xylanase activity.

AfuXyn5: The amino acid sequence of AfuXyn5 (SEQ ID NO:26) is shown inFIGS. 13B and 55B. SEQ ID NO:26 is the sequence of the immature AfuXyn5.AfuXyn5 has a predicted signal sequence corresponding to residues 1 to19 of SEQ ID NO:26 (underlined in FIG. 13B); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to residues 20 to 313 of SEQ ID NO:26. The predicted GH11conserved domains are in boldface type in FIG. 13B. AfuXyn5 was shown tohave endoxylanase activity indirectly by observing its ability tocatalyze increased xylose monomer production in the presence ofxylobiosidase when the enzymes act on pretreated biomass or on isolatedhemicellulose. The conserved catalytic residues include E119, E124, andE210. The predicted CBM is near the C-terminal end, characterized bynumerous hydrophobic residues and follows the long serine-,threonine-rich series of amino acids. The region is shown underlined inFIG. 55B. As used herein, “an AfuXyn5 polypeptide” refers to apolypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,175, 200, 250, or 275 contiguous amino acid residues among residues 20to 313 of SEQ ID NO:26. An AfuXyn5 polypeptide preferably is unaltered,as compared to native AfuXyn5, at residues E119, E120, and E210. AnAfuXyn5 polypeptide is preferably unaltered in at least 70%, 80%, 90%,95%, 98%, or 99% of the amino acid residues that are conserved amongAfuXyn5, AfuXyn2, and Trichoderma reesei Xyn2, as shown in the alignmentof FIG. 55B. An AfuXyn5 polypeptide suitably comprises the entirepredicted CBM of native AfuXyn5 and/or the entire predicted conserveddomain of native AfuXyn5 (underlined) shown in FIG. 13B. An exemplaryAfuXyn5 polypeptide comprises a sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the mature AfuXyn5 sequence shown in FIG. 13B. The AfuXyn5polypeptide of the invention preferably has xylanase activity.

Fv43D: The amino acid sequence of Fv43D (SEQ ID NO:28) is shown in FIGS.14B and 53A-53C. SEQ ID NO:28 is the sequence of the immature Fv43D.Fv43D has a predicted signal sequence corresponding to residues 1 to 20of SEQ ID NO:28 (underlined in FIG. 14B); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to residues 21 to 350 of SEQ ID NO:28. The predictedconserved domain is in boldface type in FIG. 14B. Fv43D was shown tohave β-xylosidase activity in, for example, an enzymatic assay usingp-nitophenyl-β-xylopyranoside, xylobiose, and/or mixed, linearxylo-oligomers as substrates. The predicted catalytic residues includeeither D37 or D72, D159, and E251. As used herein, “an Fv43Dpolypeptide” refers to a polypeptide and/or a variant thereof comprisinga sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50,75, 100, 125, 150, 175, 200, 250, 300, or 320 contiguous amino acidresidues among residues 21 to 350 of SEQ ID NO:28. An Fv43D polypeptidepreferably is unaltered, as compared to native Fv43D, at residues D37 orD72, D159, and E251. An Fv43D polypeptide is preferably unaltered in atleast 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues thatare conserved among a group of enzymes including Fv43D and 1, 2, 3, 4,5, 6, 7, 8, or all 9 other amino acid sequences in the alignment ofFIGS. 53A-53C. An Fv43D polypeptide suitably comprises the entirepredicted CD of native Fv43D shown in FIG. 14B. An exemplary Fv43Dpolypeptide comprises a sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the mature Fv43D sequence shown in FIG. 14B. The Fv43D polypeptide ofthe invention preferably has β-xylosidase activity.

Accordingly an Fv43D polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or(iv) 107-350 of SEQ ID NO:28. The polypeptide suitably has β-xylosidaseactivity.

Pf43B: The amino acid sequence of Pf43B (SEQ ID NO:30) is shown in FIGS.15B and 53A-53C. SEQ ID NO:30 is the sequence of the immature Pf43B.Pf43B has a predicted signal sequence corresponding to residues 1 to 20of SEQ ID NO:30 (underlined in FIG. 15B); cleavage of the signalsequence is predicted to yield a mature protein having a sequencecorresponding to residues 21 to 321 of SEQ ID NO:30. The predictedconserved domain is in boldface type in FIG. 15B. Conserved acidicresidues within the conserved domain include D32, D61, D148, and E212.Pf43B was shown to have β-xylosidase activity in, for example, anenzymatic assay using p-nitrophenyl-β-xylopyranoside, xylobiose, and/ormixed, linear xylo-oligomers as substrates. As used herein, “a Pf43Bpolypeptide” refers to a polypeptide and/or a variant thereof comprisinga sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50,75, 100, 125, 150, 175, 200, 250, or 280 contiguous amino acid residuesamong residues 21 to 321 of SEQ ID NO:30. A Pf43B polypeptide preferablyis unaltered, as compared to native Pf43B, at residues D32, D61, D148,and E212. A Pf43B polypeptide is preferably unaltered in at least 70%,80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conservedamong a group of enzymes including Pf43B and 1, 2, 3, 4, 5, 6, 7, 8, orall 9 other amino acid sequences in the alignment of FIGS. 53A-53C. APf43B polypeptide suitably comprises the predicted conserved domain ofnative Pf43B shown in FIG. 15B. An exemplary Pf43B polypeptide comprisesa sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pf43Bsequence shown in FIG. 15B. The Pf43B polypeptide of the inventionpreferably has β-xylosidase activity.

Accordingly a Pf43B polypeptide of the invention suitably comprises anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:30. The polypeptide suitably has β-xylosidase activity.

Fv51A: The amino acid sequence of Fv51A (SEQ ID NO:32) is shown in FIGS.16B and 54. SEQ ID NO:32 is the sequence of the immature Fv51A. Fv51Ahas a predicted signal sequence corresponding to residues 1 to 19 of SEQID NO:32 (underlined in FIG. 16B); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 20 to 660 of SEQ ID NO:32. The predictedL-α-arabinofuranosidase conserved domain is in boldface type in FIG.16B. Fv51A was shown to have L-α-arabinofuranosidase activity in, forexample, an enzymatic assay using 4-nitrophenyl-□α-L-arabinofuranosideas a substrate. Fv51A was shown to catalyze the release of arabinosefrom the set of oligomers released from hemicellulose via the action ofendoxylanase. Conserved residues include E42, D49, E247, E286, E330,E359, E479, and E487. As used herein, “an Fv51A polypeptide” refers to apolypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 625 contiguousamino acid residues among residues 20 to 660 of

SEQ ID NO:32. An Fv51A polypeptide preferably is unaltered, as comparedto native Fv51A, at residues E42, D49, E247, E286, E330, E359, E479, andE487. An Fv51A polypeptide is preferably unaltered in at least 70%, 80%,90%, 95%, 98%, or 99% of the amino acid residues that are conservedamong Fv51A, Pa51A, and Pf51A, as shown in the alignment of FIG. 54. AnFv51A polypeptide suitably comprises the predicted conserved domain ofnative Fv51A shown in FIG. 16B. An exemplary Fv51A polypeptide comprisesa sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv51Asequence shown in FIG. 16B. The Fv51A polypeptide of the inventionpreferably has L-α-arabinofuranosidase activity.

Accordingly an Fv51A polypeptide of the invention suitably comprise anamino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO:32, or to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or(iv) 450-660 of SEQ ID NO:32. The polypeptide suitably hasL-α-arabinofuranosidase activity.

Xvn3: The amino acid sequence of Trichoderma reesei Xyn3 (SEQ ID NO:42)is shown in FIG. 21B. SEQ ID NO:42 is the sequence of the immatureTrichoderma reesei Xyn3. Trichoderma reesei Xyn3 has a predicted signalsequence corresponding to residues 1 to 16 of SEQ ID NO:42 (underlinedin FIG. 21B); cleavage of the signal sequence is predicted to yield amature protein having a sequence corresponding to residues 17 to 347 ofSEQ ID NO:42. The predicted conserved domain is in boldface type in FIG.21B. Trichoderma reesei Xyn3 was shown to have endoxylanase activityindirectly by oberservation of its ability to catalyze increased xylosemonomer production in the presence of xylobiosidase when the enzymes acton pretreated biomass or on isolated hemicellulose. The conservedcatalytic residues include E91, E176, E180, E195, and E282, asdetermined by alignment with another GH10 family enzyme, the Xys1 deltafrom Streptomyces halstedii (Canals et al., 2003, Act Crystalogr. DBiol. 59:1447-53), which has 33% sequence identity to Trichoderma reeseiXyn3. As used herein, “a Trichoderma reesei Xyn3 polypeptide” refers toa polypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,175, 200, 250, or 300 contiguous amino acid residues among residues 17to 347 of SEQ ID NO:42. A Trichoderma reesei Xyn3 polypeptide preferablyis unaltered, as compared to native Trichoderma reesei Xyn3, at residuesE91, E176, E180, E195, and E282. A Trichoderma reesei Xyn3 polypeptideis preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% ofthe amino acid residues that are conserved between Trichoderma reeseiXyn3 and Xys1 delta. A Trichoderma reesei Xyn3 polypeptide suitablycomprises the entire predicted conserved domain of native Trichodermareesei Xyn3 shown in FIG. 21B. An exemplary Trichoderma reesei Xyn3polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the mature Trichoderma reesei Xyn3 sequence shown in FIG. 21B. TheTrichoderma reesei Xyn3 polypetpide of the invention preferably hasxylanase activity.

Xvn2: The amino acid sequence of Trichoderma reesei Xyn2 (SEQ ID NO:43)is shown in FIGS. 22 and 55B. SEQ ID NO:43 is the sequence of theimmature Trichoderma reesei Xyn2. Trichoderma reesei Xyn2 has apredicted prepropeptide sequence corresponding to residues 1 to 33 ofSEQ ID NO:43 (underlined in FIG. 22); cleavage of the predicted signalsequence between positions 16 and 17 is predicted to yield a propeptide,which is processed by a kexin-like protease between positions 32 and 33,generating the mature protein having a sequence corresponding toresidues 33 to 222 of SEQ ID NO:43. The predicted conserved domain is inboldface type in FIG. 22. Trichoderma reesei Xyn2 was shown to haveendoxylanase activity indirectly by observation of its ability tocatalyze an increased xylose monomer production in the presence ofxylobiosidase when the enzymes act on pretreated biomass or on isolatedhemicellulose. The conserved acidic residues include E118, E123, andE209. As used herein, “a Trichoderma reesei Xyn2 polypeptide” refers toa polypeptide and/or a variant thereof comprising a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150,or 175contiguous amino acid residues among residues 33 to 222 of SEQ IDNO:43. A Trichoderma reesei Xyn2 polypeptide preferably is unaltered, ascompared to a native Trichoderma reesei Xyn2, at residues E118, E123,and E209. A Trichoderma reesei Xyn2 polypeptide is preferably unalteredin at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residuesthat are conserved among Trichoderma reesei Xyn2, AfuXyn2, and AfuXyn5,as shown in the alignment of FIG. 55B. A Trichoderma reesei Xyn2polypeptide suitably comprises the entire predicted conserved domain ofnative Trichoderma reesei Xyn2 shown in FIG. 22. An exemplaryTrichoderma reesei Xyn2 polypeptide comprises a sequence having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to the mature Trichoderma reesei Xyn2 sequenceshown in FIG. 22. The Trichoderma reesei Xyn2 polypeptide of theinvention preferably has xylanase activity.

Bxl1: The amino acid sequence of Trichoderma reesei Bxl1 (SEQ ID NO:44)is shown in FIGS. 23 and 64. SEQ ID NO:44 is the sequence of theimmature Trichoderma reesei Bxl1. Trichoderma reesei Bxl1 has apredicted signal sequence corresponding to residues 1 to 18 of SEQ IDNO:44 (underlined in FIG. 23); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 19 to 797 of SEQ ID NO:44. The predicted conserved domains arein boldface type in FIG. 23. Trichoderma reesei Bxl1 was shown to haveβ-xylosidase activity in, for example, an enzymatic assay usingp-nitophenyl-β-xylopyranoside, xylobiose and/or mixed, linearxylo-oligomers as substrates. The conserved acidic residues includeE193, E234, and D310. As used herein, “a Trichoderma reesei Bxl1polypeptide” refers to a polypeptide and/or a variant thereof comprisinga sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, or 750 contiguous amino acid residues among residues 17 to 797of SEQ ID NO:44. A Trichoderma reesei Bxl1 polypeptide preferably isunaltered, as compared to a native Trichoderma reesei Bxl1, at residuesE193, E234, and D310. A Trichoderma reesei Bxl1 polypeptide ispreferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of theamino acid residues that are conserved among Trichoderma reesei Bxl1,Fv3A, and Trichoderma reesei Bgl1, as shown in the alignment of FIGS.64A-64B. A Trichoderma reesei Bxl1 polypeptide suitably comprises theentire predicted conserved domains of native Trichoderma reesei Bxl1shown in FIG. 23. An exemplary Trichoderma reesei Bxl1 polypeptidecomprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the matureTrichoderma reesei Bxl1 sequence shown in FIG. 23. The Trichodermareesei Bxl1 polypeptide of the invention preferably has β-xylosidaseactivity.

Accordingly a Trichoderma reesei Bxl1 polypeptide of the inventionsuitably comprises an amino acid sequence with at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 44. The polypeptide suitably hasβ-xylosidase activity.

Bgl1: The amino acid sequence of Trichoderma reesei Bgl1 (SEQ ID NO:45)is shown in FIGS. 24 and 64A-64B. Trichoderma reesei Bgl1 has apredicted signal sequence corresponding to residues 1 to 19 of SEQ IDNO:45 (underlined in FIG. 24); cleavage of the signal sequence ispredicted to yield a mature protein having a sequence corresponding toresidues 20 to 744 of SEQ ID NO:45. The predicted conserved domain is inboldface type in FIG. 24. Trichoderma reesei Bgl1 has been shown to haveβ-glucosidase activity by observation of a capacity to catalyze thehydrolysis of para-nitrophenyl-β-D-glucopyranoside to produce para-nitrophenol, and a capacity to catalyze the hydrolysis of cellobiose.The conserved acidic residues include D164, E197, and D267. As usedherein, “a Trichoderma reesei Bgl1 polypeptide” refers to a polypeptideand/or a variant thereof comprising a sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, or 780 contiguous aminoacid residues among residues 20 to 744 of SEQ ID NO:45. A Trichodermareesei Bgl1 polypeptide preferably is unaltered, as compared to a nativeBgl1, at residues D164, E197, and D267. A Trichoderma reesei Bgl1polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%,or 99% of the amino acid residues that are conserved among Trichodermareesei Bgl1, Fv3A, and Trichoderma reesei Bxl1, as shown in thealignment of FIGS. 64A-64B. A Trichoderma reesei Bgl1 polypeptidesuitably comprises the entire predicted conserved domain of nativeTrichoderma reesei Bgl1 shown in FIG. 24. An exemplary Trichodermareesei Bgl1 polypeptide comprises a sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the mature Trichoderma reesei Bgl1 sequence shown in FIG.24. The Trichoderma reesei Bgl1 polypeptide of the invention preferablyhas β-glucosidase activity.

Accordingly, the present disclosure provides a number of isolated,synthetic, or recombinant hemicelluloytic polypeptides or variants asdescribed below:

-   (1) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    24 to 766 of SEQ ID NO:2; (ii) 73 to 321 of SEQ ID NO:2; (iii) 73 to    394 of SEQ ID NO:2; (iv) 395 to 622 of SEQ ID NO:2; (v) 24 to 622 of    SEQ ID NO:2; or (iv) 73 to 622 of SEQ ID NO:2; the polypeptide    preferably has β-xylosidase activity; or-   (2) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 445 of SEQ ID NO:4; (ii) 21 to 301 of SEQ ID NO:4; (iii) 21 to    323 of SEQ ID NO:4; (iv) 21 to 444 of SEQ ID NO:4; (v) 302 to 444 of    SEQ ID NO:4; (vi) 302 to 445 of SEQ ID NO:4; (vii) 324 to 444 of SEQ    ID NO:4; or (viii) 324 to 445 of SEQ ID NO:4; the polypeptide    preferably has β-xylosidase activity; or-   (3) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    19 to 530 of SEQ ID NO:6; (ii) 29 to 530 of SEQ ID NO:6; (iii) 19 to    300 of SEQ ID NO:6; or (iv) 29 to 300 of SEQ ID NO:6; the    polypeptide preferably has β-xylosidase activity; or-   (4) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    20 to 439 of SEQ ID NO:8; (ii) 20 to 291 of SEQ ID NO:8; (iii) 145    to 291 of SEQ ID NO:8; or (iv) 145 to 439 of SEQ ID NO:8; the    polypeptide preferably has β-xylosidase activity; or-   (5) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    23 to 449 of SEQ ID NO:10; (ii) 23 to 302 of SEQ ID NO:10; (iii) 23    to 320 of SEQ ID NO:10; (iv) 23 to 448 of SEQ ID NO:10; (v) 303 to    448 of SEQ ID NO:10; (vi) 303 to 449 of SEQ ID NO:10; (vii) 321 to    448 of SEQ ID NO:10; or (viii) 321 to 449 of SEQ ID NO:10; the    polypeptide preferably has β-xylosidase activity; or-   (6) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    17 to 574 of SEQ ID NO:12; (ii) 27 to 574 of SEQ ID NO:12; (iii) 17    to 303 of SEQ ID NO:12; or (iv) 27 to 303 of SEQ ID NO:12; the    polypeptide preferably has both β-xylosidase activity and    L-α-arabinofuranosidase activity; or-   (7) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 676 of SEQ ID NO:14; (ii) 21 to 652 of SEQ ID NO:14; (iii) 469    to 652 of SEQ ID NO:14; or (iv) 469 to 676 of SEQ ID NO:14; the    polypeptide preferably has both β-xylosidase activity and    L-α-arabinofuranosidase activity; or-   (8) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    19 to 340 of SEQ ID NO:16; (ii) 53 to 340 of SEQ ID NO:16; (iii) 19    to 383 of SEQ ID NO:16; or (iv) 53 to 383 of SEQ ID NO:16; the    polypeptide preferably has β-xylosidase activity; or-   (9) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 341 of SEQ ID NO:18; (ii) 107 to 341 of SEQ ID NO:18; (iii) 21    to 348 of SEQ ID NO:18; or (iv) 107 to 348 of SEQ ID NO:18; the    polypeptide preferably has β-xylosidase activity; or-   (10) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    15 to 558 of SEQ ID NO:20; or (ii) 15 to 295 of SEQ ID NO:20; the    polypeptide preferably has L-α-arabinofuranosidase activity; or-   (11) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 632 of SEQ ID NO:22; (ii) 461 to 632 of SEQ ID NO:22; (iii) 21    to 642 of SEQ ID NO:22; or (iv) 461 to 642 of SEQ ID NO:22; the    polypeptide preferably has L-α-arabinofuranosidase activity; or-   (12) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    20 to 341 of SEQ ID NO:28; (ii) 21 to 350 of SEQ ID NO:28; (iii) 107    to 341 of SEQ ID NO:28; or (iv) 107 to 350 of SEQ ID NO:28; the    polypeptide has β-xylosidase activity; or-   (13) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 660 of SEQ ID NO:32; (ii) 21 to 645 of SEQ ID NO:32; (iii) 450    to 645 of SEQ ID NO:32; or (iv) 450 to 660 of SEQ ID NO:32; the    polypeptide preferably has L-α-arabinofuranosidase activity.

The present disclosure provides also compositions (e.g., cellulasecompositions, or enzyme blends/compositions) or fermentation brothsenriched with one or more of the above-described polypeptides. Theenzyme blend/composition is thus a non-naturally-occurring composition.The cellulase composition can be, for example, a filamentous fungalcellulase composition, such as a Trichoderma cellulase composition.

The fermentation broth can be a fermentation broth of a filamentousfungus, for example, a Trichoderma, Humicola, Fusarium, Aspergillus,Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia,Mucor, Cochliobolus, Pyricularia, or Chrysosporium fermentation broth.In particular, the fermentation broth can be, for example, one ofTrichoderma spp. such as a Trichoderma reesei, or Penicillium spp., suchas a Penicillium funiculosum. The fermentation broth can also suitablybe a cell-free fermentation broth.

Additionally the instant disclosure provides host cells that arerecombiantly engineered to express a polypeptide described above. Thehost cells can be, for example, filamentous fungal host cells, such asTrichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, cochliobolus,Pyricularia, or Chrysosporium cells. In particular, the host cells canbe, for example, a Trichoderma spp. cell (such as a Trichoderma reeseicell), or a Penicillium cell (such as a Penicillium funiculosum cell),an Aspergillus cell (such as an Aspergillus oryzae or Aspergillusnidulans cell), or a Fusarium cell (such as a Fusarium verticilloides orFusarium oxysporum cell).

6.1.1 Fusion Proteins

The present disclosure also provides a fusion protein that includes adomain of a protein of the present disclosure attached to one or morefusion segments, which are typically heterologous to the protein (i.e.,derived from a different source than the protein of the disclosure).Suitable fusion segments include, without limitation, segments that canenhance a protein's stability, provide other desirable biologicalactivity, and/or facilitate purification of the protein (e.g., byaffinity chromatography). A suitable fusion segment can be a domain ofany size that has the desired function (e.g., imparts increasedstability, solubility, action or biological activity; and/or simplifiespurification of a protein). Fusion segments can be joined to aminoand/or carboxyl termini of the domain(s) of a protein of the presentdisclosure. The fusion segments can be susceptible to cleavage. Theremay be some advantage in having this susceptibility, for example, it mayenable straight-forward recovery of the protein of interest. Fusionproteins are preferably produced by culturing a recombinant celltransfected with a fusion nucleic acid that encodes a protein, whichincludes a fusion segment attached to either the carboxyl or aminoterminal end, or fusion segments attached to both the carboxyl and aminoterminal ends, of a protein, or a domain thereof.

Accordingly, proteins of the present disclosure also include expressionproducts of gene fusions (e.g., an overexpressed, soluble, and activeform of a recombinant protein), of mutagenized genes (e.g., genes havingcodon modifications to enhance gene transcription and translation), andof truncated genes (e.g., genes having signal sequences removed orsubstituted with a heterologous signal sequence).

Glycosyl hydrolases that utilize insoluble substrates are often modularenzymes. They usually comprise catalytic modules appended to one or morenon-catalytic carbohydrate-binding domains (CBMs). In nature, CBMs arethought to promote the glycosyl hydrolase's interaction with its targetsubstrate polysaccharide. Thus, the disclosure provides chimeric enzymeshaving altered substrate specificity;

including, for example, chimeric enzymes having multiple substrates as aresult of “spliced-in” heterologous CBMs. The heterologous CBMs of thechimeric enzymes of the disclosure can also be designed to be modular,such that they are appended to a catalytic module or catalytic domain (a“CD”, e.g., at an active site), which can likewise be heterologous orhomologous to the glycosyl hydrolase.

Thus, the disclosure provides peptides and polypeptides consisting of,or comprising, CBM/CD modules, which can be homologously paired orjoined to form chimeric (heterologous) CBM/CD pairs. Thus, thesechimeric polypeptides/peptides can be used to improve or alter theperformance of an enzyme of interest. Accordingly, the disclosureprovides chimeric enzymes comprising, e.g., at least one CBM of anenzyme of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 43, 44, or 45. A polypeptide of thedisclosure, for example, includes an amino acid sequence comprising theCD and/or CBM of the glycosyl hydrolase sequence of SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,43, 44, or 45. The polypeptide of the disclosure can thus suitably be afusion protein comprising functional domains from two or more differentproteins (e.g., a CBM from one protein linked to a CD from anotherprotein).

The polypeptides of the disclosure can suitably be obtained and/or usedin “substantially pure” form. For example, a polypeptide of thedisclosure constitutes at least about 80 wt. % (e.g., at least about 85wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96wt. %, 97 wt. %, 98 wt. %, or 99 wt. %) of the total protein in a givencomposition, which also includes other ingredients such as a buffer orsolution.

Also, the polypeptides of the disclosure can suitably be obtained and/orused in recombinant culture broths (e.g., a filamentous fungal culturebroth). The recombinant culture broths can be non-naturally occurring;for example, the culture broth can be produced by a recombinant hostcell that is engineered to express a heterologous polypeptide of thedisclosure, or by a recombinant host cell that is engineered to expressan endogenous polypeptide of the disclosure in greater or lesser amountsthan the endogenous expression levels (e.g., in an amount that is 1-,2-, 3-, 4-, 5-, or more- fold greater or less than the endogenousexpression levels). Furthermore, the polypeptides of the disclosure cansuitably be obtained and/or used as recombinant culture broths producedby “integrated” host cell strains that have been engineered to express aplurality of polypeptides of the disclosure in desired ratios. Exemplarydesired ratios are described herein, for example, in Section 6.3.4below.

6.2 Nucleic Acids and Host Cells

The present disclosure provides nucleic acids encoding a polypeptide ofthe disclosure, for example one described in Section 6.1 above.

The disclosure provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid sequence having at least about 70%, e.g., atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, or complete (100%) sequence identity to a nucleic acidof SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at leastabout 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or2000 nucleotides. The present disclosure also provides nucleic acidsencoding at least one polypeptide having a hemicellulolytic activity(e.g., a xylanase, β-xylosidase, and/or L-α-arabinofuranosidaseactivity).

Nucleic acids of the disclosure also include isolated, synthetic orrecombinant nucleic acids encoding an enzyme having the sequence of SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 43, 44, or 45, and subsequences thereof (e.g., aconserved domain or carbohydrate binding domain (“CBM”), and variantsthereof. A nucleic acid of the disclosure can, for example, encode themature portion of a protein of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45.

The disclosure specifically provides a nucleic acid encoding an Fv3A, aPf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, anFo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, a Fv43D, a Pf43B, anFv43B, an Fv51A, a Trichoderma reesei Xyn3, a Trichoderma reesei Xyn2, aTrichoderma reesei Bxl1, or a Trichoderma reesei Bgl1 polypeptide.

For example, the disclosure provides an isolated nucleic acid molecule,wherein the nucleic acid molecule encodes:

-   (1) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    24 to 766 of SEQ ID NO:2; (ii) 73 to 321 of SEQ ID NO:2; (iii) 73 to    394 of SEQ ID NO:2; (iv) 395 to 622 of SEQ ID NO:2; (v) 24 to 622 of    SEQ ID NO:2; or (iv) 73 to 622 of SEQ ID NO:2; or-   (2) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 445 of SEQ ID NO:4; (ii) 21 to 301 of SEQ ID NO:4; (iii) 21 to    323 of SEQ ID NO:4; (iv) 21 to 444 of SEQ ID NO:4; (v) 302 to 444 of    SEQ ID NO:4; (vi) 302 to 445 of SEQ ID NO:4; (vii) 324 to 444 of SEQ    ID NO:4; or (viii) 324 to 445 of SEQ ID NO:4; or-   (3) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    19 to 530 of SEQ ID NO:6; (ii) 29 to 530 of SEQ ID NO:6; (iii) 19 to    300 of SEQ ID NO:6; or (iv) 29 to 300 of SEQ ID NO:6; or-   (4) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    20 to 439 of SEQ ID NO:8; (ii) 20 to 291 of SEQ ID NO:8; (iii) 145    to 291 of SEQ ID NO:8; or (iv) 145 to 439 of SEQ ID NO:8; or-   (5) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    23 to 449 of SEQ ID NO:10; (ii) 23 to 302 of SEQ ID NO:10; (iii) 23    to 320 of SEQ ID NO:10; (iv) 23 to 448 of SEQ ID NO:10; (v) 303 to    448 of SEQ ID NO:10; (vi) 303 to 449 of SEQ ID NO:10; (vii) 321 to    448 of SEQ ID NO:10; or (viii) 321 to 449 of SEQ ID NO:10; or-   (6) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    17 to 574 of SEQ ID NO:12; (ii) 27 to 574 of SEQ ID NO:12; (iii) 17    to 303 of SEQ ID NO:12; or (iv) 27 to 303 of SEQ ID NO:12; or-   (7) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 676 of SEQ ID NO:14; (ii) 21 to 652 of SEQ ID NO:14; (iii) 469    to 652 of SEQ ID NO:14; or (iv) 469 to 676 of SEQ ID NO:14; or-   (8) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    19 to 340 of SEQ ID NO:16; (ii) 53 to 340 of SEQ ID NO:16; (iii) 19    to 383 of SEQ ID NO:16; or (iv) 53 to 383 of SEQ ID NO:16; or-   (9) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 341 of SEQ ID NO:18; (ii) 107 to 341 of SEQ ID NO:18; (iii) 21    to 348 of SEQ ID NO:18; or (iv) 107 to 348 of SEQ ID NO:18; or-   (10) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    15 to 558 of SEQ ID NO:20; or (ii) 15 to 295 of SEQ ID NO:20; or-   (11) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 632 of SEQ ID NO:22; (ii) 461 to 632 of SEQ ID NO:22; (iii) 21    to 642 of SEQ ID NO:22; or (iv) 461 to 642 of SEQ ID NO:22; or-   (12) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    20 to 341 of SEQ ID NO:28; (ii) 21 to 350 of SEQ ID NO:28; (iii) 107    to 341 of SEQ ID NO:28; or (iv) 107 to 350 of SEQ ID NO:28; or-   (13) a polypeptide comprising an amino acid sequence with at least    90%, at least 95%, at least 98%, at least 99%, or 100% sequence    identity to the amino acid sequence corresponding to positions (i)    21 to 660 of SEQ ID NO:32; (ii) 21 to 645 of SEQ ID NO:32; (iii) 450    to 645 of SEQ ID NO:32; or (iv) 450 to 660 of SEQ ID NO:32.

The instant disclosure also provides:

-   (1) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:1, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:1, or to a    fragment thereof; or-   (2) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:3, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:3, or to a    fragment thereof; or-   (3) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:5, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:5, or to a    fragment thereof; or-   (4) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:7, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:7, or to a    fragment thereof; or-   (5) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:9, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:9, or to a    fragment thereof; or-   (6) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:11, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:11, or to a    fragment thereof; or-   (7) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:13, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:13, or to a    fragment thereof; or-   (8) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:15, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:15, or to a    fragment thereof; or-   (9) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:17, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:17, or to a    fragment thereof; or-   (10) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:19, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:19, or to a    fragment thereof; or-   (11) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:21, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:21, or to a    fragment thereof; or-   (12) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:27, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:27, or to a    fragment thereof; or-   (13) a nucleic acid having at least 90% (e.g., at least 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to    SEQ ID NO:31, or a nucleic acid that is capable of hybridizing under    high stringency conditions to a complement of SEQ ID NO:31, or to a    fragment thereof.

The disclosure also provides expression cassettes and/or vectorscomprising the above-described nucleic acids.

Suitably, the nucleic acid encoding an enzyme of the disclosure isoperably linked to a promoter. Specifically, where recombinantexpression in a filamentous fungal host is desired, the promoter can bea filamentous fungal promoter. The nucleic acids can be, for example,under the control of heterologous promoters. The nucleic acids can alsobe expressed under the control of constitutive or inducible promoters.Examples of promoters that can be used include, but are not limited to,a cellulase promoter, a xylanase promoter, the 1818 promoter (previouslyidentified as a highly expressed protein by EST mapping Trichoderma).For example, the promoter can suitably be a cellobiohydrolase,endoglucanase, or β-glucosidase promoter. A particulary suitablepromoter can be, for example, a T. reesei cellobiohydrolase,endoglucanase, or β-glucosidase promoter. For example, the promoter is acellobiohydrolase I (cbh1) promoter. Non-limiting examples of promotersinclude a cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, pki1, gpd1, xyn1, orxyn2 promoter. Additional non-limiting examples of promoters include aT. reesei cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, pki1, gpd1, xyn1, orxyn2 promoter.

The present disclosure provides host cells that are engineered toexpress one or more enzymes of the disclosure. Suitable host cellsinclude cells of any microorganism (e.g., cells of a bacterium, aprotist, an alga, a fungus (e.g., a yeast or filamentous fungus), orother microbe), and are preferably cells of a bacterium, a yeast, or afilamentous fungus.

Suitable host cells of the bacterial genera include, but are not limitedto, cells of Escherichia, Bacillus, Lactobacillus, Pseudomonas, andStreptomyces. Suitable cells of bacterial species include, but are notlimited to, cells of Escherichia coli, Bacillus subtilis, Bacilluslicheniformis, Lactobacillus brevis, Pseudomonas aeruginosa, andStreptomyces lividans.

Suitable host cells of the genera of yeast include, but are not limitedto, cells of Saccharomyces, Schizosaccharomyces, Candida, Hansenula,Pichia, Kluyveromyces, and Phaffia. Suitable cells of yeast speciesinclude, but are not limited to, cells of Saccharomyces cerevisiae,Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha,Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffiarhodozyma.

Suitable host cells of filamentous fungi include all filamentous formsof the subdivision Eumycotina. Suitable cells of filamentous fungalgenera include, but are not limited to, cells of Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium,Coprinus, Coriolus, Corynascus, Chaetomium, Cryptococcus, Filobasidium,Fusarium, Gibberella, Humicola, Hypocrea, Magnaporthe, Mucor,Myceliophthora, Mucor, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Scytaldium,Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trametes, and Trichoderma. Suitable cells can alsoinclude cells of various anamorph and teleomorph forms fo thesefilamentous fungal genera.

Suitable cells of filamentous fungal species include, but are notlimited to, cells of Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta,Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinuscinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa,Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurosporaintermedia, Penicillium purpurogenum, Penicillium canescens, Penicilliumsolitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phiebiaradiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma llongibrachiatum, Trichoderma reesei,and Trichoderma viride.

The disclosure further provides a recombinant host cell that isengineered to express one or more, two or more, three or more, four ormore, or five or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A,an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, anAfuXyn5 an Fv43D, a Pf43B, and an Fv51A polypeptide. The recombinanthost cell is, for example, a recombinant Trichoderma reesei host cell.In a particular example, the disclosure provides a recombinant fungus,such as a recombinant Trichoderma reesei, that is engineered to expressone or more, two or more, three or more, four or more, or five or moreof an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, aGz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, aPf43B, and an Fv51A polypeptide. The disclosure provides a recombinantTrichoderma reesei host cell engineered to express 1, 2, 3, 4, 5, ormore of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, aPa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, anFv43D, a Pf43B, and an Fv51A polypeptide.

The disclosure provides a host cell, for example, a recombinant fungalhost cell or a recombinant filamentous fungus, engineered torecombinantly express at least one xylanase, at least one β-xylosidase,and one L-α-arabinofuranosidase. The disclosure also provides arecombinant host cell , e.g., a recombinant fungal host cell or arecombinant filamentous fungus such as a recombinant Trichoderma reesei,that is engineered to express 1, 2, 3, 4, 5, or more of an Fv3A, aPf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, anFo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, andan Fv51A polypeptide, in addition to one or more of Trichoderma reeseiXyn2, Trichoderma reesei Xyn3, Trichoderma reesei Bxl1 and/orTrichoderma reesei Bgl1. The recombinant host cell is, for example, aTrichoderma reesei host cell. The recombinant fungus is, for example, arecombinant Trichoderma reesei. The disclosure provides a Trichodermareesei host cell, or a recombinant Trichoderma reesei fungus, that isengineered to recombinantly express 1, 2, 3, 4, 5, or more of an Fv3A, aPf43A, an Fv43E, an Fv39A, an Fv43A,an Fv43B, a Pa51A, a Gz43A, anFo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, andan Fv51A polypeptide, in addition to recombiantly express one or more ofTrichoderma reesei Xyn2, Trichoderma reesei Xyn3, Trichoderma reeseiBxl1 and/or Trichoderma reesei Bgl1.

The present disclosure also provides a recombinant host cell e.g., arecombinant fungal host cell or a recombinant organism, e.g., afilamentous fungus, such as a recombinant Trichoderma reesei, that isengineered to recombinantly express Trichoderma reesei Xyn3, Trichodermareesei Bgl1, Fv3A, Fv43D, and Fv51A polypeptides. For example, therecombinant host cell is suitably a Trichoderma reesei host cell. Therecombinant fungus is suitably a recombinant Trichoderma reesei. Thedisclosure provides, for example, a Trichoderma reesei host cellengineered to recombinantly express Trichoderma reesei Xyn3, Trichodermareesei Bgl1, Fv3A, Fv43D, and Fv51A polypeptides.

Additionally the disclosure provides a recombinant host cell orrecombinant fungus that is engineered to express an enzyme blendcomprising suitable enzymes in ratios suitable for saccharification. Therecombinant host cell is, for example, a fungal host cell. Therecombinant fungus is, for example, a recombinant Trichoderma reesei.Exemplary enzyme ratios/amounts present in suitable enzyme blends aredescribed in Section 6.3.4 below.

The disclosure further provides transgenic plants comprising a nucleicacid of the disclosure or an expression cassette of the disclosure. Thetransgenic plant can be, for example, a cereal plant, a corn plant, apotato plant, a tomato plant, a wheat plant, an oilseed plant, arapeseed plant, a soybean plant, a rice plant, a barley plant, or atobacco plant.

6.3 Enzyme Blends for Saccharification

The present disclosure provides a composition comprising an enzymeblend/composition that is capable of breaking down lignocellulosematerial. Such a multi-enzyme blend/composition comprises at least onepolypeptide of the present disclosure, in combination with one or moreadditional polypeptides of the present disclosure, or one or moreenzymes from other microorganisms, plants, or organisms. Synergisticenzyme combinations and related methods are contemplated. The disclosureincludes methods for identifying the optimum ratios of the enzymesincluded in the blends/compositions for degrading a particularlignocellulosic material. These methods include, e.g., tests to identifythe optimum enzyme blend/composition and ratios for efficient conversionof a given lignocellulosic substrate to its constituent sugars. TheExamples below include assays that may be used to identify optimumratios and blends/compositions of enzymes with which to degradelignocellulosic materials.

6.3.1 Background

The cell walls of higher plants are comprised of a variety ofcarbohydrate polymer (CP) components. These CP interact through covalentand non-covalent means, providing the structural integrity required toform rigid cell walls and resist turgor pressure in plants. The major CPfound in plants is cellulose, which forms the structural backbone of thecell wall. During cellulose biosynthesis, chains of poly-β-1,4-D-glucoseself associate through hydrogen bonding and hydrophobic interactions toform cellulose microfibrils, which further self-associate to form largerfibrils. Cellulose microfibrils are often irregular structurally andcontain regions of varying crystallinity. The degree of crystallinity ofcellulose fibrils depends on how tightly ordered the hydrogen bonding isbetween and among its component cellulose chains. Areas withless-ordered bonding, and therefore more accessible glucose chains, arereferred to as amorphous regions.

The general model for cellulose depolymerization to glucose involves aminimum of three distinct enzymatic activities. Endoglucanases cleavecellulose chains internally to shorter chains in a process thatincreases the number of accessible ends, which are more susceptible toexoglucanase activity than the intact cellulose chains. Theseexoglucanases (e.g., cellobiohydrolases) are specific for eitherreducing ends or non-reducing ends, liberating, in most cases,cellobiose, the dimer of glucose. . The accumulating cellobiose is thensubject to cleavage by cellobiases (e.g., β-1,4-glucosidases) toglucose.

Cellulose contains only anhydro-glucose. In contrast, hemicellulosecontains a number of different sugar monomers. For instance, aside fromglucose, sugar monomers in hemicellulose can also include xylose,mannose, galactose, rhamnose, and arabinose. Hemicelluloses mostlycontain D-pentose sugars and occasionally small amounts of L-sugars.Xylose is typically present in the largest amount, but mannuronic acidand galacturonic acid also tend to be present. Hemicelluloses includexylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan.

The enzymes and multi-enzyme compositions of the disclosure are usefulfor saccharification of hemicellulose materials, including, e.g., xylan,arabinoxylan, and xylan- or arabinoxylan-containing substrates.Arabinoxylan is a polysaccharide composed of xylose and arabinose,wherein L-α-arabinofuranose residues are attached as branch-points to aβ-(1,4)-linked xylose polymeric backbone.

Most biomass sources are rather complex, containing cellulose,hemicellulose, pectin, lignin, protein, and ash, among other components.Accordingly, in certain aspects, the present disclosure provides enzymeblends/compositions containing enzymes that impart a range or variety ofsubstrate specificities when working together to degrade biomass intofermentable sugars in the most efficient manner. One example of amulti-enzyme blend/composition of the present invention is a mixture ofcel lobiohydrolase(s), xylanase(s), endoglucanase(s), β-glucosidase(s),β-xylosidase(s), and, optionally, accessory proteins. The enzymeblend/composition is suitably a non-naturally occurring composition.

Accordingly, the disclosure provides enzyme blends/compositions(including products of manufacture) comprising a mixture ofxylan-hydrolyzing, hemicellulose- and/or cellulose-hydrolyzing enzymes,which include at least one, several, or all of a cellulase, including aglucanase; a cellobiohydrolase; an L-α-arabinofuranosidase; a xylanase;a β-glucosidase; and a β-xylosidase. Preferably each of the enzymeblends/compositions of the disclosure comprises at least one enzyme ofthe disclosure. The present disclosure also provides enzymeblends/compositions that are non-naturally occurring compositions. Asused herein, the term “enzyme blends/compositions” refers to:

-   -   (1) a composition made by combining component enzymes, whether        in the form of a fermentation broth or partially or completely        isolated or purified;    -   (2) a composition produced by an organism modified to express        one or more component enzymes; in certain embodiments, the        organism used to express one or more component enzymes can be        modified to delete one or more genes; in certain other        embodiments, the organism used to express one or more component        enzymes can further comprise proteins affecting xylan        hydrolysis, hemicellulose hydrolysis, and/or cellulose        hydrolysis;    -   (3) a composition made by combining component enzymes        simultaneously, separately, or sequentially during a        saccharification or fermentation reaction; and    -   (4) an enzyme mixture produced in situ, e.g., during a        saccharification or fermentation reaction;    -   (5) a composition produced in accordance with any or all of the        above (1)-(4).

The term “fermentation broth” as used herein refers to an enzymepreparation produced by fermentation that undergoes no or minimalrecovery and/or purification subsequent to fermentation. For example,microbial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes). Then, once the enzyme(s) are secreted into the cell culturemedia, the fermentation broths can be used. The fermentation broths ofthe disclosure can contain unfractionated or fractionated contents ofthe fermentation materials derived at the end of the fermentation. Forexample, the fermentation broths of the invention are unfractionated andcomprise the spent culture medium and cell debris present after themicrobial cells (e.g., filamentous fungal cells) undergo a fermentationprocess. The fermentation broth can suitably contain the spent cellculture media, extracellular enzymes, and live or killed microbialcells. Alternatively, the fermentation broths can be fractionated toremove the microbial cells. In those cases, the fermentation broths can,for example, comprise the spent cell culture media and the extracellularenzymes.

Any of the enzymes described specifically herein can be combined withany one or more of the enzymes described herein or with any otheravailable and suitable enzymes, to produce a suitable multi-enzymeblend/composition. The disclosure is not restricted or limited to thespecific exemplary combinations listed below.

6.3.2 Biomass

The disclosure provides methods and processes for biomasssaccharification, using enzymes, enzyme blends/compositions of thedisclosure. The term “biomass,” as used herein, refers to anycomposition comprising cellulose and/or hemicellulose (optionally alsolignin in lignocellulosic biomass materials). As used herein, biomassincludes, without limitation, seeds, grains, tubers, plant waste orbyproducts of food processing or industrial processing (e.g., stalks),corn (including, e.g., cobs, stover, and the like), grasses (including,e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g.,Panicum species, such as Panicum virgatum), wood (including, e.g., woodchips, processing waste), paper, pulp, and recycled paper (including,e.g., newspaper, printer paper, and the like). Other biomass materialsinclude, without limitation, potatoes, soybean (e.g., rapeseed), barley,rye, oats, wheat, beets, and sugar cane bagasse.

The disclosure provides methods of saccharification comprisingcontacting a composition comprising a biomass material, for example, amaterial comprising xylan, hemicellulose, cellulose, and/or afermentable sugar, with a polypeptide of the disclosure, or apolypeptide encoded by a nucleic acid of the disclosure, or any one ofthe enzyme blends/compositions, or products of manufacture of thedisclosure.

The saccharified biomass (e.g., lignocellulosic material processed byenzymes of the disclosure) can be made into a number of bio-basedproducts, via processes such as, e.g., microbial fermentation and/orchemical synthesis. As used herein, “microbial fermentation” refers to aprocess of growing and harvesting fermenting microorganisms undersuitable conditions. The fermenting microorganism can be anymicroorganism suitable for use in a desired fermentation process for theproduction of bio-based products. Suitable fermenting microorganismsinclude, without limitation, filamentous fungi, yeast, and bacteria. Thesaccharified biomass can, for example, be made it into a fuel (e.g., abiofuel such as a bioethanol, biobutanol, biomethanol, a biopropanol, abiodiesel, a jet fuel, or the like) via fermentation and/or chemicalsynthesis. The saccharified biomass can, for example, also be made intoa commodity chemical (e.g., ascorbic acid, isoprene, 1,3-propanediol),lipids, amino acids, proteins, and enzymes, via fermentation and/orchemical synthesis.

6.3.3. Pretreatment

Prior to saccharification, biomass (e.g., lignocellulosic material) ispreferably subject to one or more pretreatment step(s) in order torender xylan, hemicellulose, cellulose and/or lignin material moreaccessible or susceptable to enzymes and thus more amenable tohydrolysis by the enzyme(s) and/or enzyme blends/compositions of thedisclosure.

In an exemplary embodiment, the pretreatment entails subjecting biomassmaterial to a catalyst comprising a dilute solution of a strong acid anda metal salt in a reactor. The biomass material can, e.g., be a rawmaterial or a dried material. This pretreatment can lower the activationenergy, or the temperature, of cellulose hydrolysis, ultimately allowinghigher yields of fermentable sugars. See, e.g., U.S. Pat. Nos.6,660,506; 6,423,145.

Another exemplary pretreatment method entails hydrolyzing biomass bysubjecting the biomass material to a first hydrolysis step in an aqueousmedium at a temperature and a pressure chosen to effectuate primarilydepolymerization of hemicellulose without achieving significantdepolymerization of cellulose into glucose. This step yields a slurry inwhich the liquid aqueous phase contains dissolved monosaccharidesresulting from depolymerization of hemicellulose, and a solid phasecontaining cellulose and lignin. The slurry is then subject to a secondhydrolysis step under conditions that allow a major portion of thecellulose to be depolymerized, yielding a liquid aqueous phasecontaining dissolved/soluble depolymerization products of cellulose.See, e.g., U.S. Pat. No. 5,536,325.

A further exemplary method involves processing a biomass material by oneor more stages of dilute acid hydrolysis using about 0.4% to about 2% ofa strong acid; followed by treating the unreacted solid lignocellulosiccomponent of the acid hydrolyzed material with alkaline delignification.See, e.g., U.S. Pat. No. 6,409,841.

Another exemplary pretreatment method comprises prehydrolyzing biomass(e.g., lignocellulosic materials) in a prehydrolysis reactor; adding anacidic liquid to the solid lignocellulosic material to make a mixture;heating the mixture to reaction temperature; maintaining reactiontemperature for a period of time sufficient to fractionate thelignocellulosic material into a solubilized portion containing at leastabout 20% of the lignin from the lignocellulosic material, and a solidfraction containing cellulose; separating the solubilized portion fromthe solid fraction, and removing the solubilized portion while at ornear reaction temperature; and recovering the solubilized portion. Thecellulose in the solid fraction is rendered more amenable to enzymaticdigestion. See, e.g., U.S. Pat. No. 5,705,369.

Further pretreatment methods can involve the use of hydrogen peroxideH₂O₂. See Gould, 1984, Biotech, and Bioengr. 26:46-52.

Pretreatment can also comprise contacting a biomass material withstoichiometric amounts of sodium hydroxide and ammonium hydroxide at avery low concentration. See Teixeira et al.,1999, Appl. Biochem.andBiotech. 77-79:19-34.

Pretreatment can also comprise contacting a lignocellulose with achemical (e.g., a base, such as sodium carbonate or potassium hydroxide)at a pH of about 9 to about 14 at moderate temperature, pressure, andpH. See PCT Publication WO2004/081185.

Ammonia is used, for example, in a preferred pretreatment method. Such apretreatment method comprises subjecting a biomass material to lowammonia concentration under conditions of high solids. See, e.g., U.S.Patent Publication No. 20070031918 and PCT publication WO 06110901.

6.3.4 Exemplary Enzyme Blends

The present disclosure provides enzyme blends/compositions comprisingone or more enzymes of the disclosure. One or more enzymes of the enzymeblends/compositions can be produced by a recombinant host cell or arecombinant organism. The enzyme blends/compositions are suitablynon-naturally occurring compositions.

An enzyme blend/composition of the disclosure can suitably comprise afirst polypeptide having β-xylosidase activity, and further comprises 1,2, 3, or 4 of a second polypeptide having β-xylosidase activity, one ormore polypeptides having L-α-arabinofuranosidase activity, one or morepolypeptides having xylanase activity, and one or more polypeptideshaving cellulase activity. The first polypeptide having β-xylosidaseactivity is, for example, an Fv3A, a Pf43A, an Fv43E, an Fv43A, anFv43B, a Pa51A, a Gz43A, an Fo43A, or an Fv39A polypeptide. The secondpolypeptide having β-xylosidase activity, if present, is, for example,different from the first polypeptide having β-xylosidase activity, andis suitably an Fv3A, a Pf43A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, aGz43A, an Fv43D, an Fo43A, an Fv39A, or a Trichoderma reesei Bxl1polypeptide. Each of the one or more polypeptides havingL-α-arabinofuranosidase activity, if present, is, for example, an Af43A,a Pf51A, a Pa51A, an Fv43B, or an Fv51A polypeptide. Each of the one ormore polypeptides having xylanase activity is, for example, aTrichoderma reesei Xyn, a Trichoderma reesei Xyn2, an AfuXyn2, or anAfuXyn5. Each of the one or more polypeptides having cellulase activity,if present, is, for example, an endoglucanase, for example, aTrichoderma reesei EG1 or EG2, a cellobiohydrolase, for example, aTrichoderma reesei CBH1 or CBH2, or a β-glucosidase, for example, aTrichoderma reesei Bgl1.

Another enzyme blend/composition of the disclosure can suitably comprisea first polypeptide having L-α-arabinofuranosidase activity, and furthercomprises 1, 2, 3, or 4 of a second polypeptide havingL-α-arabinofuranosidase activity, one or more polypeptides havingβ-xylosidase activity, one or more polypeptides having xylanaseactivity, and/or one or more polypeptides having cellulase activity. Thefirst L-α-arabinofuranosidase is an Af43A, a Pf51A, or an Fv51Apolypeptide. The second L-α-arabinofuranosidase is different from thefirst L-α-arabinofuranosidase, and is, for example, an Af43A, a Pf51A, aPa51A, an Fv43B, or an Fv51A polypeptide. Each of the one or moreβ-xylosidases is, for example, an Fv3A, a Pf43A, an Fv43E, an Fv43A, anFv43B, a Pa51A, an Fv43D, a Gz43A, an Fo43A, an Fv39A, or a Trichodermareesei Bxl1 polypeptide. In certain embodiments, each of the one or morexylanases is a Trichoderma reesei Xyn3, a Trichoderma reesei Xyn2, anAfuXyn2, or an AfuXyn5 polypeptide. In certain embodiments, each of theone or more cellulases is independently an endoglucanase, for example, aTrichoderma reesei EG1 or EG2 polypeptide, a cellobiohydrolase, forexample, a Trichoderma reesei CBH1 or CBH2 polypeptide, or aβ-glucosidase, for example, a Trichoderma reesei Bgl1 polypeptide.

X lanases: The xylanase(s) suitably constitutes about 0.05 wt. % toabout 75 wt. % of the enzymes in an enzyme blend/ composition of thedisclosure (i.e., the percentage xylanase(s) is a weight percentagerelative to the weight of all proteins in the composition) or a relativeweight basis (i.e., wherein the percentage xylanase(s) is a weightpercentage relative to the combined weight of xylanases, β-xylosidases,cellulases, L-α-arabinofuranosidases, and accessory proteins). The ratioof any pair of proteins relative to each other can be readily calculatedin the enzyme blends/compositions of the disclosure. Blends/compositionscomprising enzymes in any weight ratio derivable from the weightpercentages disclosed herein are contemplated. The xylanase content canbe in a range wherein the lower limit is 0.05 wt. %, 1 wt. %, 2 wt. %, 3wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %,12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 40 wt. %, 45 wt. %, or50 wt. % of the total weight of enzymes in the enzyme blend/composition,and the upper limit is 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %,35 wt. %, 40 wt. %, 50 wt. %, 55 wt. %, 60 wt. %, 65 wt. %, 70 wt. %, or75 wt. % of the total weight of enzymes in the enzyme blend/composition.The one or more xylanases in an enzyme blend or composition canrepresent, for example, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, or 15wt. % to 25 wt. % of the total enzymes in the enzyme blend/composition.Exemplary suitable xylanases for inclusion in the enzymeblends/compositions of the disclosure are described in Section 6.3.6below.

L-α-arabinofuranosidases: The L-α-arabinofuranosidase(s) suitablyconstitutes about 0.05 wt. % to about 75 wt. % of the total weight ofall enzymes in a given enzyme blend/composition (i.e., wherein thepercentage L-α-arabinofuranosidase(s) is a weight percentage relative tothe weight of all proteins in the blend/composition) or a relativeweight basis (i.e., wherein the percentage L-α-arabinofuranosidase(s) isa weight percentage relative to the combined weight of xylanases,β-xylosidases, cellulases, L-α-arabinofuranosidases, and accessoryproteins). The ratio of any pair of proteins relative to each other canbe readily calculated based on the disclosure. Blends/compositionscomprising enzymes in any weight ratio derivable from the weightpercentages disclosed herein are contemplated. TheL-α-arabinofuranosidase content can be in a range wherein the lowerlimit is 0.05 wt. %, 1 wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt.% 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25wt. %, 30 wt. %, 40 wt. %, 45 wt. %, or 50 wt. % of the total weight ofenzymes in the blend/composition, and the upper limit is 10 wt. %, 15wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 55wt. %, 60 wt. %, 65 wt. %, 70 wt. % or 75 wt. % of the total weight ofenzymes in the blend/composition. For example, the one or moreL-α-arabinofuranosidase(s) can suitably represent 2 wt. % to 25 wt. %; 5wt. % to 20 wt. %; or 5 wt. % to 10 wt. % of the total weight of enzymesin the blend/composition. Exemplary suitable L-α-arabinofuranosidase(s)for inclusion in the enzyme blends/compositions of the disclosure aredescribed in Section 6.3.8 below.

β-Xylosidases: The β-xylosidase(s) suitably constitutes about 0.05 wt. %to about 75 wt. % of the total weight of enzymes in an enzymeblend/composition. The ratio of any pair of proteins relative to eachother can be readily calculated based on the disclosure herein. Blends/compositions comprising enzymes in any weight ratio derivable from theweight percentages disclosed herein are contemplated. The β-xylosidasecontent can be in a range wherein the lower limit is about 0.05 wt. %, 1wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. % 7 wt. %, 8 wt. %, 9wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 40wt. %, 45 wt. %, or 50 wt. % of the total weight of enzymes in theblend/composition, and the upper limit is about 10 wt,%, 15 wt,%, 20 wt.%, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 55 wt. %, 60 wt. %,65 wt. %, 70 wt. %, or 75 wt. % of the total weight of enzymes in theblend/composition. For example, the β-xylosidase(s) can represent about0.05 wt. % to about 75 wt. % of the total weight of enzymes in theblend/composition. Also, the β-xylosidase(s) can represent 0.05 wt. % toabout 70 wt. %, about 1 wt. % to about 65 wt. %, about 1 wt. % to about60 wt. %, about 2 wt. % to about 55 wt%, about 3 wt. % to about 50 wt.%, about 4 wt. % to about 45 wt. %, or about 5 wt. % to about 40 wt. %of the total weight of enzymes in the blend/composition. In yet afurther example, the β-xylosidase(s) suitably represent 2 wt. % to 30wt. %; 10 wt. % to 20 wt. %; or 5 wt. % to 10 wt. % of the total weightof enzymes in the blend/composition. Exemplary suitable β-xylosidase(s)are described in Section 6.3.7 below.

Cellulases: The cellulase(s) suitably constitutes about 0.05 wt. % toabout 90 wt. % of the total weight of enzymes in an enzymeblend/composition. Ratio of any pair of proteins relative to each othercan be readily calculated based on the disclosure herein.Blends/compositions comprising enzymes in any weight ratio derivablefrom the weight percentages disclosed herein are contemplated. Thecellulase content can be in a range wherein the lower limit is about0.05 wt. %, 5 wt. %, 10 wt. %, 20 wt. %, 30 wt. %, 40 wt. %, 50 wt. %,60 wt. %, 70 wt. % of the total weight of enzymes in theblend/composition, and the upper limit is about 20 wt. %, 30 wt. %, 40wt. %, 50 wt. %, 60 wt. %, 70 wt. %, 80 wt. %, or 90 wt. % of the totalweight of enzymes in the blend/composition. For example, thecellulase(s) suitably represents 30 wt. % to 80 wt. %, 50 wt. % to 70wt. %, or 40 wt. % to 60 wt. % of the total weight of enzymes in theblend/composition. Exemplary suitable cellulases are described inSection 6.3.5 below. The cellulase components in an enzymeblend/composition of the disclosure are suitably capable of achieving atleast about 0.005 fraction product per mg protein per gram of phosphoricacid swollen cellulose (PASO) as determined by a calcofluor assay. Forexample, the cellulase components in a blend/composition of thedisclosure are capable of achieving a range of fraction product per mgprotein per gram of PASO, wherein the lower limit of the range is about0.005, 0.01, 0.015, 0.02, 0.03, 0.04, 0.05, 0.06, 0.075, or 0.1, andwherein the upper limit of the range is, 0.03, 0.04, 0.05, 0.06, 0.075,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7. The cellulase components in ablend/composition of the disclosure can, for example, achieve0.00005-0.0001, 0.0005 -0.001, 0.001- 0.005, 0.005- 0.03, 0.01-0.06,0.02-0.04, 0.01-0.03, 0.02-0.05, 0.02-0.04, 0.01-0.05, 0.015-0.035, or0.015-0.075 product fraction product per mg protein per gram PASO asdetermined by a calcofluor assay. The cellulase can be, for example, awhole cellulase. The cellulase can also, for example, suitably beenriched with a β-glucosidase.

Accessory Proteins: The enzyme blend/composition may suitably furthercomprise one or more accessory proteins. The accessory protein contentof an enzyme blend/composition can range from about 0 wt. % to about 60wt. % of the total weight of proteins in an enzyme blend/composition.Ratio of any pair of proteins relative to each other can be readilydetermined based on the disclosure herein. Blends/compositionscomprising enzymes in any weight ratio derivable from the weightpercentages disclosed herein are contemplated. The accessory proteincontent can be in a range wherein the lower limit is 0 wt. %, 1 wt. %, 2wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 10 wt. %,12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, or 35 wt. % of the total weightof proteins in the enzyme blend/composition, and the upper limit is 2wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. % 15 wt. %, 20 wt. %, 30 wt.%, 40 wt. %, 50 wt. %, or 60 wt. % of the total weight of proteins inthe enzyme blend/composition. For example, the accessory protein(s) cansuitably represent 0 wt. % to 2 wt. %, 5 wt. % to 10 wt. %, 20 wt. % to50 wt. %, or 2 wt. % to 5 wt. % in the enzyme blend/composition.Exemplary suitable accessory proteins for inclusion in the enzymeblends/compositions of the disclosure are described in Section 6.3.9below.

The present disclosure provides a first enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 30 wt. % to about 80 wt. % (e.g., 30 wt. % to 80 wt.        %, 35 wt. % to 75 wt. %, 40 wt. % to 70 wt. %, 40 wt. % to 60        wt. %, 50 wt. % to 70 wt. %, etc.) cellulase(s), e.g., whole        cellulase or β-glucosidase enriched whole cellulase;    -   (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %,        10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %,        15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma        reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5,        or a mixture of two or more of the foregoing enzymes;    -   (3) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %,        5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %,        10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.)        β-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an        Fv43E, an Fv43A, an Fv43B, a Pa51A, an Fo43A, a Gz43A, a        Trichoderma reesei Bxl1, or a mixture of two or more of the        foregoing enzymes;    -   (4) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %,        5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 25 wt. %,        5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc)        L-α-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a        Pf51A, an Fv51A, or a mixture of two or more of the foregoing        enzymes; and    -   (5) about 0 wt. % to about 50 wt. % (2 wt. % to 40 wt. %, 5 wt.        % to 30 wt. %, 10 wt. % to 25 wt. %, 0 wt. % to 2 wt. %, 5 wt. %        to 10 wt. %, 20 wt. % to 50 wt. %, 2 wt. % to 5 wt. %, etc)        accessory protein(s).

The present disclosure provides a second enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 30 wt. % to about 80 wt. % (e.g., 30 wt. % to 80 wt.        %, 35 wt. % to 75 wt. %, 40 wt. % to 70 wt. %, 40 wt. % to 60        wt. %, 50 wt. % to 70 wt. %, etc.) cellulase(s), e.g., whole        cellulase or β-glucosidase enriched whole cellulase, or about 2        wt. % to about 10 wt. % (e.g., 2 wt. % to 8 wt. %, 4 wt. % to 6        wt. %, 2 wt. % to 4 wt. %, 6 wt. % to 8 wt. %, 8 wt. % to 10 wt.        %, 2 wt. % to 10 wt. %, etc) β-glucosidase(s), e.g., a        Trichoderma reesei Bgl1;    -   (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %,        10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %,        15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma        reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5,        or a mixture of two or more of the foregoing enzymes;    -   (3) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %,        5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %,        10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) of at least two        β-xylosidase(s), wherein at least one β-xylosidase is selected        from Group 1 and at least one β-xylosidase is selected from        Group 2;    -   wherein:        -   Group 1: an Fv3A, an Fv43A, or a mixture thereof;        -   Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei            Bxl1, a Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a            mixture of two or more of the foregoing enzymes;    -   (4) about 2 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %,        5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc)        L-α-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a        Pf51A, Fv51A, or a mixture of two or more of the foregoing        enzymes; and    -   (5) about 0 wt. % to about 50 wt. % (2 wt. % to 40 wt. %, 5 wt.        % to 30 wt. %, 10 wt. % to 25 wt. %, 0 wt. % to 2 wt. %, 5 wt. %        to 10 wt. %, 20 wt. % to 50 wt. %, 2 wt. % to 5 wt. %, etc)        accessory protein(s).

The present disclosure provides a third enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %,        10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %,        15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma        reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5,        or a mixture of two or more of the foregoing enzymes; and    -   (2) about 2 wt. % to 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt.        % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt.        % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) of at least two        β-xylosidase(s), wherein at least one β-xylosidase is selected        from Group 1 and at least one β-xylosidase is selected from        Group 2;    -   wherein:        -   Group 1: an Fv3A, an Fv43A, or a mixture thereof;        -   Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei            Bxl1, a

Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a mixture of two ormore of the foregoing enzymes.

The present disclosure provides a fourth enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 5 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %,        5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) xylanase(s),        e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an        AfuXyn2, an AfuXyn5, or a mixture of two or more of the        foregoing enzymes;    -   (2) about 2 wt. % to about 30 wt. % (e.g., 2 wt. % to 30 wt. %,        10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.)        β-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an        Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, a        Trichoderma reesei Bxl1, or a mixture of two or more of the        foregoing enzymes; and    -   (3) about 2 wt. % to about 50 wt. % (e.g., 2 wt. % to 5 wt. %, 5        wt. % to 45 wt. %, 10 wt. % to 40 wt. %, 15 wt. % to 30 wt. %,        10 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 15 wt. % to 40 wt. %,        etc) β-glucosidase(s), e.g., a Trichoderma reesei Bgl1.

The present disclosure provides a fifth enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 5 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %,        5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) xylanase(s),        e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an        AfuXyn2, an AfuXyn5, or a mixture of two or more of the        foregoing enzymes; and    -   (2) about 2 wt. % to about 30 wt. % (e.g., 2 wt. % to 30 wt. %,        10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.)        β-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an        Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, a        Trichoderma reesei Bxl1, or a mixture of two or more of the        foregoing enzymes.

The present disclosure provides a sixth enzyme blend/composition forlignocellulose saccharification comprising:

-   -   (1) about 2 wt. % to about 50 wt. % (e.g., 2 wt. % to 5 wt. %, 5        wt. % to 45 wt. %, 10 wt. % to 40 wt. %, 15 wt. % to 30 wt. %,        10 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 15 wt. % to 40 wt. %,        etc) β-glucosidase(s), e.g., a Bgl1;    -   (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %,        10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %,        15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma        reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5,        or a mixture of two or more of the foregoing enzymes;    -   (3) about 2 wt. % to 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt.        % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt.        % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) β-xylosidase(s), e.g.,        an Fv3A, a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fv43A, an        Fv43B, a Pa51A, a Gz43A, an Fo43A, a Trichoderma reesei Bxl1, or        a mixture of two or more of the foregoing enzymes; and    -   (4) about 2 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %,        5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc)        L-α-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a        Pf51A, Fv51A, or a mixture of two or more of the foregoing        enzymes.

The sixth enzyme blend/composition for lignocellulose saccharificationabove can, for example, comprise about 2 wt. % to about 40 wt. % of atleast two β-xylosidase, wherein at least one β-xylosidase is selectedfrom Group 1 and at least one β-xylosidase is selected from Group 2;wherein:

-   -   Group 1: an Fv3A, an Fv43A, or a mixture thereof;    -   Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei Bxl1,        a Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a mixture of        two or more of the foregoing enzymes.

Where an enzyme blend/composition of the disclosure contains both Group1 and a Group 2 β-xylosidases, the ratio of Group 1 to Group 2β-xylosidases is preferably 1:10 to 10:1. For example, the ratio issuitably 1:2 to 2:1, 2:5 to 5:2, 3:8 to 8:3, 1:4 to 4:1, 1:5 to 5:1, 1:7to 7:1, or any range between any pair the foregoing endpoints (e.g.,1:10 to 2:1, 4:1 to 2:5, 3:8 to 5:1, etc.). A particular example of asuitable ratio is approximately 1:1.

Where an enzyme blend/composition of the disclosure contains an Fv43A asa β-xylosidase, the blend/composition can further contain Fv43B as anL-α-arabinofuranosidase.

An enzyme blend/composition of the disclosure is, for example, suitablypart of a saccharification reaction mixture containing biomass inaddition to the components of the enzyme blend/composition. For example,the saccharification reaction mixture can be characterized by 1, 2, 3 orall 4 of the following features:

-   -   (1) the total weight of xylanase(s) per kg of hemicellulase in        said saccharification reaction mixture is in a range in which        the lower limit is about 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 7 g, or        10 g, and the upper limit is independently about 5 g, 7 g, 10 g,        15 g, 20 g, 30 g, or 40 g; for example, the total weight of        xylanase(s) per kg of hemicellulase in the reaction mixture can        be 0.5 g to 40 g, 0.5g to 30 g, 0.5 g to 20 g, 0.5 to 10g, 0.5        to 5g, 1 g to 40 g, 2 g to 40g, 3 g to 40 g, 5 g to 40 g, 7 g to        30 g, 10 g to 30 g, 5 gto20 g, or 5 g to 30 g.    -   (ii) the total weight of β-xylosidase(s) per kg of hemicellulase        in said saccharification reaction mixture is in a range in which        the lower limit is about 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 7 g, or        10 g and the upper limit is independently about 5 g, 7 g, 10 g,        15 g, 20 g, 30 g, 40 g, or 50 g; for example, the total weight        of β-xylosidase(s) per kg of hemicellulase in the reaction        mixture can be 0.5 g to 40 g, 0.5 to 50 g, 0.5 g to 30 g, 0.5 g        to 20 g, 0.5 g to 10 g, 0.5 g to 5g, 1 g to 40 g, 2 g to 40 g, 3        g to 40 g, 5 gto 40g, 7 g to 30 g, 10 g to 30 g, 5 g to 30 g, 5        g to20 g.    -   (iii) the total weight of L-α-arabinofuranosidase(s) per kg of        hemicellulase in said saccharification reaction mixture is in a        range in which the lower limit is about 0.2 g, 0.5 g, 1 g, 1.5        g, 2 g, 2.5 g, 3 g, 4 g, or 5 g and the upper limit is        independently about 2 g, 3 g, 4 g, 5 g, 7 g, 10 g, 15 g, or 20        g; for example, the total weight of L-α-arabinofuranosidase(s)        per kg of hemicellulase in the reaction mixture can be 0.2 g to        20 g, 0.5 g to 20 g, 1 g to 20 g, 2 g to 20 g, 2.5 g to 20 g, 3        g to 15 g, 4 g to 20 g, 5 g to 15 g, 5 g to 10g, 5 g to 20 g, or        2.5 g to 15 g.    -   (iv) the total weight of cellulase(s) per kg of cellulase in        said saccharification reaction mixture is in a range in which        the lower limit is about 1 g, 3 g, 5 g, 7 g, 10 g, 12 g, 15 g,        18 g, or 20g, and the upper limit is independently about 10 g,        15 g, 18 g, 20 g, 25 g, 30g, 50 g, 75 g, or 100 g; for example,        the total weight of cellulase(s) per kg of cellulase in said        reaction mixture can be 1 g to 100 g, 3 g to 100 g, 5 g to 100        g, 7 g to 100 g, 12 g to 100 g, 15g to 100g, 18 gto 100g, 3 g to        75 g, 5 g to 50 g, 7 g to 75 g, 10 gto 75g, 10 g to 50 g, 12 g        to 75 g, 12 g to 50 g, 15 g to 75 g, 15 g to 50 g, 18 g to 30 g,        18 g to 75g.

6.3.5. Cellulases

The enzyme blends/compositions of the disclosure can comprise one ormore cellulases. Cellulases are enzymes that hydrolyze cellulose(β-1,4-glucan or β D-glucosidic linkages) resulting in the formation ofglucose, cellobiose, cellooligosaccharides, and the like. Cellulaseshave been traditionally divided into three major classes: endoglucanases(EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91)(“CBH”) and β-glucosidases (β-D-glucoside glucohydrolase; EC 3.2.1.21)(“BG”) (Knowles et al., 1987, Trends in Biotechnology 5(9):255-261;Shulein, 1988, Methods in Enzymology, 160:234-242). Endoglucanases actmainly on the amorphous parts of the cellulose fiber, whereascellobiohydrolases are also able to degrade crystalline cellulose.

Cellulases for use in accordance with the methods and compositions ofthe disclosure can be obtained from, or produced recombinantly from,inter alia, one or more of the following organisms: Crinipellisscapella, Macrophomina phaseolina, Myceliophthora thermophila, Sordariafimicola, Volutella colletotrichoides, Thielavia terrestris, Acremoniumsp., Exidia glandulosa, Fomes fomentarius, Spongipellis sp.,Rhizophlyctis rosea, Rhizomucor pusillus, Phycomyces niteus,Chaetosty/um fresenii, Diplodia gossypina, Ulospora bilgramii,Saccobolus dilutellus, Penicillium verrucu/osum, Penicilliumchrysogenum, Thermomyces verrucosus, Diaporthe syngenesia,Colletotrichum lagenarium, Nigrospora sp., Xylaria hypoxylon, Nectriapinea, Sordaria macrospora, Thielavia thermophila, Chaetomium mororum,Chaetomium virscens, Chaetomium brasiliensis, Chaetomium cunicolorum,Syspastospora boninensis, Cladorrhinum foecundissimum, Scytalidiumthermophila, Gliocladium catenulatum, Fusarium oxysporum ssp.lycopersici, Fusarium oxysporum ssp. passiflora, Fusarium solani,Fusarium anguioides, Fusarium poae, Humicola nigrescens, Humicolagrisea, Panaeolus retirugis, Trametes sanguinea, Schizophyllum commune,Trichothecium roseum, Microsphaeropsis sp., Acsobolus stictoideus spej.,Poronia punctata, Nodulisporum sp., Trichoderma sp. (e.g., Trichodermareesei) and Cylindrocarpon sp.

For example, a cellulase for use in the method and/or composition of thedisclosure is a whole cellulase and/or is capable of achieving at least0.1 (e.g. 0.1 to 0.4) fraction product as determined by the calcofluorassay described in Section 7.1.10 below.

6.3.5.1 β-Glucosidase

The enzyme blends/compositions of the disclosure can optionally compriseone or more β-glucosidases. The term “β-glucosidase” as used hereinrefers to a β-D-glucoside glucohydrolase classified as EC 3.2.1.21,and/or members of certain GH families, including, without limitation,members of GH families 1, 3, 9 or 48, which catalyze the hydrolysis ofcellobiose to release β-D-glucose.

Suitable β-glucosidase can be obtained from a number of microorganisms,by recombinant means, or be purchased from commercial sources. Examplesof β-glucosidases from microorganisms include, without limitation, onesfrom bacteria and fungi. For example, a β-glucosidase of the presentdisclosure is suitably obtained from a filamentous fungus.

The β-glucosidases can be obtained, or produced recombinantly, from,inter alia, Aspergillus aculeatus (Kawaguchi et al. Gene 1996, 173:287-288), Aspergillus kawachi (Iwashita et al. Appl. Environ. Microbiol.1999, 65: 5546-5553), Aspergillus oryzae (WO 2002/095014), Cellulomonasbiazotea (Wong et al. Gene, 1998, 207:79-86), Penicillium funiculosum(WO 2004/078919), Saccharomycopsis fibuligera (Machida et al. Appl.Environ. Microbiol. 1988, 54: 3147-3155), Schizosaccharomyces pombe(Wood et al. Nature 2002, 415: 871-880), or Trichoderma reesei (e.g.,β-glucosidase 1 (U.S. Pat. No. 6,022,725), β-glucosidase 3 (U.S. Pat.No.6,982,159), β-glucosidase 4 (U.S. Pat. No. 7,045,332), β-glucosidase5 (U.S. Pat. No. 7,005,289), β-glucosidase 6 (U.S. Publication No.20060258554), β-glucosidase 7 (U.S. Publication No. 20060258554)).

The β-glucosidase can be produced by expressing an endogenous orexogenous gene encoding a β-glucosidase. For example, β-glucosidase canbe secreted into the extracellular space e.g., by Gram-positiveorganisms (e.g., Bacillus or Actinomycetes), or eukaryotic hosts (e.g.,Trichoderma, Aspergillus, Saccharomyces, or Pichia). The β-glucosidasecan be, in some circumstances, overexpressed or underexpressed.

The β-glucosidase can also be obtained from commercial sources. Examplesof commercial β-glucosidase preparation suitable for use in the presentdisclosure include, for example, Trichoderma reesei β-glucosidase inAccellerase® BG (Danisco US Inc.,

Genencor); NOVOZYM™ 188 (a β-glucosidase from Aspergillus niger);Agrobacterium sp. β-glucosidase, and Thermatoga maritima β-glucosidasefrom Megazyme (Megazyme International Ireland Ltd., Ireland.).

Moreover, the β-glucosidase can be a component of a whole cellulase, asdescribed in Section 6.3.5.4 below.

β-glucosidase activity can be determined by a number of suitable meansknown in the art, such as the assay described by Chen et al., inBiochimica et Biophysica Acta 1992, 121:54-60, wherein 1 pNPG denotes 1μmoL of Nitrophenol liberated from 4-nitrophenyl-β-D-glucopyranoside in10 min at 50° C. (122° F.) and pH 4.8.

6.3.5.2 Endocilucanases

The enzyme blends/compositions of the disclosure optionally comprise oneor more endoglucanase. Any endoglucanase (EC 3.2.1.4) can be used in themethods and compositions of the present disclosure. An endoglucanse canbe produced by expressing an endogenous or exogenous endoglucanase gene.The endoglucanase can be, in some circumstances, overexpressed orunderexpressed.

For example, Trichoderma reesei EG1 (Penttila et al., Gene 1986,63:103-112) and/or EG2 (Saloheimo et al., Gene 1988, 63:11-21) aresuitably used in the methods and compositions of the present disclosure.

A thermostable Thielavia terrestris endoglucanase (Kvesitadaze et al.,Applied Biochem. Biotech. 1995, 50:137-143) is, in another example, usedin the methods and compositions of the present disclosure. Moreover, aTrichoderma reesei EG3 (Okada et al. Appl. Environ. Microbiol. 1988,64:555-563), EG4 (Saloheimo et al. Eur. J. Biochem. 1997, 249:584-591),EG5 (Saloheimo et al. Molecular Microbiology 1994, 13:219-228), EG6(U.S. Patent Publication No. 20070213249), or EG7 (U.S. PatentPublication No.

20090170181), an Acidothermus cellulolyticus El endoglucanase (U.S. Pat.No. 5,536,655), a Humicola insolens endoglucanase V (EGV) (Protein DataBank entry 4ENG), a Staphylotrichum coccosporum endoglucanase (U.S.Patent Publication No. 20070111278), an Aspergillus aculeatusendoglucanase F1-CMC (Ooi et al. Nucleic Acid Res. 1990, 18:5884), anAspergillus kawachii IFO 4308 endoglucanase CMCase-1 (Sakamoto et al.Curr. Genet. 1995, 27:435-439), an Erwinia carotovara (Saarilahti et al.Gene 1990, 90:9-14); or an Acremonium thermophilum ALK04245endoglucanase (U.S. Patent Publication No. 20070148732) can also beused. Additional suitable endoglucanases are described in, e.g., WO91/17243, WO 91/17244, WO 91/10732, U.S. Pat. No. 6,001,639.

6.3.5.3 Cellobiohydrolases

Any cellobiohydrolase (EC 3.2.1.91) (“CBH”) can be optionally used inthe methods and blends/compositions of the present disclosure. Thecellobiohydrolase can be produced by expressing an endogeneous orexogeneous cellobiohydrolase gene. The cellobiohydrolase can be, in somecircumstances, overexpressed or under expressed.

For example, Trichoderma reesei CBHI (Shoemaker et al. Bio/Technology1983, 1:691-696) and/or CBHII (Teeri et al. Bio/Technology 1983,1:696-699) can be suitably used in the methods and blends/compositionsof the present disclosure.

Suitable CBHs can be selected from an Agaricus bisporus CBH1 (Swiss ProtAccession No. Q92400), an Aspergillus aculeatus CBH1 (Swiss ProtAccession No.

059843), an Aspergillus nidulans CBHA (GenBank Accession No. AF420019)or CBHB (GenBank Accession No. AF420020), an Aspergillus niger CBHA(GenBank Accession No. AF156268) or CBHB (GenBank Accession No.AF156269), a Claviceps purpurea CBH1 (Swiss Prot Accession No. 000082),a Cochliobolus carbonarum CBH1 (Swiss Prot Accession No. Q00328), aCryphonectria parasitica CBH1 (Swiss Prot Accession No. Q00548), aFusarium oxysporum CBH1 (Cel7A) (Swiss Prot Accession No. P46238), aHumicola grisea CBH1.2 (GenBank Accession No. U50594), a Humicola griseavar. thermoidea CBH1 (GenBank Accession No. D63515) a CBHI.2 (GenBankAccession No. AF123441), or an exo1 (GenBank Accession No. AB003105), aMelanocarpus albomyces Cel7B (GenBank Accession No. AJ515705), aNeurospora crassa CBHI (GenBank Accession No. X77778), a Penicilliumfuniculosum CBHI (Cel7A) (U.S. Patent Publication No. 20070148730), aPenicillium janthinellum CBHI (GenBank Accession No. S56178), aPhanerochaete chrysosporium CBH (GenBank Accession No. M22220), or aCBHI-2 (Cel7D) (GenBank Accession No. L22656), a Talaromyces emersoniiCBH1A (GenBank Accession No. AF439935), a Trichoderma viride CBH1(GenBank Accession No. X53931), or a Volvariella volvacea V14 CBH1(GenBank Accession No. AF156693).

6.3.5.4 Whole Cellulases

An enzyme blend/composition of the disclosure can further comprise awhole cellulase. As used herein, a “whole cellulase” refers to either anaturally occurring or a non-naturally occurring cellulase-containingcomposition comprising at least 3 different enzyme types: (1) anendoglucanase, (2) a cellobiohydrolase, and (3) a β-glucosidase, orcomprising at least 3 different enzymatic activities: (1) anendoglucanase activity, which catalyzes the cleavage of internal β-1,4linkages, resulting in shorter glucooligosaccharides, (2) acellobiohydrolase activity, which catalyzes an “exo”-type release ofcellobiose units (β-1,4 glucose-glucose disaccharide), and (3) aβ-glucosidase activity, which catalyzes the release of glucose monomerfrom short cel looligosaccharides (e.g., cellobiose).

A “naturally occurring cellulase-containing” composition is one producedby a naturally occurring source, which comprises one or morecellobiohydrolase-type, one or more endoglucanase- type, and one or moreβ-glucosidase-type components or activities, wherein each of thesecomponents or activities is found at the ratio and level produced innature, untouched by the human hand. Accordingly, a naturally occurringcellulase-containing composition is, for example, one that is producedby an organism unmodified with respect to the cellulolytic enzymes suchthat the ratio or levels of the component enzymes are unaltered fromthat produced by the native organism in nature. A “non-naturallyoccurring cellulase-containing composition” refers to a compositionproduced by: (1) combining component cellulolytic enzymes either in anaturally occurring ratio or a non-naturally occurring, i.e., altered,ratio; or (2) modifying an organism to overexpress or underexpress oneor more cellulolytic enzymes; or (3) modifying an organism such that atleast one cellulolytic enzyme is deleted. A “non-naturally occurringcellulase containing” composition can also refer to a compositionresulting from adjusting the culture conditions for anaturally-occurring organism, such that the naturally-occurring organismgrows under a non-native condition, and produces an altered level orratio of enzymes. Accordingly, in some embodiments, the whole cellulasepreparation of the present disclosure can have one or more EGs and/orCBHs and/or β-glucosidases deleted and/or overexpressed.

In the present disclosure, a whole cellulase preparation can be from anymicroorganism that is capable of hydrolyzing a cellulosic material. Insome embodiments, the whole cellulase preparation is a filamentousfungal whole cellulase. For example, the whole cellulase preparation canbe from an Acremonium, Aspergillus, Emericella, Fusarium, Humicola,Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, or Trichoderma species. The whole cellulase preparationis, for example, an Aspergillus aculeatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, or Aspergillus oryzae whole cellulase. Moreover, thewhole cellulase preparation can be a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum whole cellulase preparation. The whole cellulase preparationcan also be a Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Penicillium funiculosum, Scytalidium thermophilum, or Thielaviaterrestris whole cellulase preparation. Moreover, the whole cellulasepreparation can be a Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei (e.g., RL-P37(Sheir-Neiss G et al. Appl. Microbiol. Biotechnology, 1984, 20,pp.46-53), QM9414 (ATCC No. 26921), NRRL 15709, ATCC 13631, 56764,56466, 56767), or a Trichoderma viride (e.g., ATCC 32098 and 32086)whole cellulase preparation.

The whole cellulase preparation can, in particular, suitably be aTrichoderma reesei RutC30 whole cellulase preparation, which isavailable from the American Type Culture Collection as Trichodermareesei ATCC 56765. For example, the whole cellulase preparation can alsosuitably be a whole cellulase of Penicillium funiculosum, which isavailable from the American Type Culture Collection as Penicilliumfuniculosum ATCC Number: 10446.

The whole cellulase preparation can also be obtained from commercialsources. Examples of commercial cellulase preparations suitable for usein the methods and compositions of the present disclosure include, forexample, CELLUCLAST™ and Cellic™ (Novozymes A/S) and LAMINEX™ BG,IndiAge™ 44L, Primafast™ 100, Primafast™ 200, Spezyme™ CP, Accellerase®1000 and Accellerase® 1500 (Danisco US. Inc., Genencor).

Suitable whole cellulase preparations can be made using anymicroorganism cultivation methods known in the art, especiallyfermentation, resulting in the expression of enzymes capable ofhydrolyzing a cellulosic material. As used herein, “fermentation” refersto shake flask cultivation, small- or large-scale fermentation, such ascontinuous, batch, fed-batch, or solid state fermentations in laboratoryor industrial fermenters performed in a suitable medium and underconditions that allow the cellulase and/or enzymes of interest to beexpressed and/or isolated.

Generally, the microorganism is cultivated in a cell culture mediumsuitable for production of enzymes capable of hydrolyzing a cellulosicmaterial. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures and variations known in the art. Suitable culture media,temperature ranges and other conditions for growth and cellulaseproduction are known in the art. As a non-limiting example, a typicaltemperature range for the production of cellulases by Trichoderma reeseiis 24° C. to 28° C.

The whole cellulase preparation can be used as it is produced byfermentation with no or minimal recovery and/or purification. Forexample, once cellulases are secreted into the cell culture medium, thecell culture medium containing the cellulases can be used directly. Thewhole cellulase preparation can comprise the unfractionated contents offermentation material, including the spent cell culture medium,extracellular enzymes and cells. On the other hand, the whole cellulasepreparation can also be subject to further processing in a number ofroutine steps, e.g., precipitation, centrifugation, affinitychromatography, filtration, or the like. For example, the wholecellulase preparation can be concentrated, and then used without furtherpurification. The whole cellulase preparation can, for example, beformulated to comprise certain chemical agents that decrease cellviability or kills the cells after fermentation. The cells can, forexample, be lysed or permeabilized using methods known in the art.

The endoglucanase activity of the whole cellulase preparation can bedetermined using carboxymethyl cellulose (CMC) as a substrate. Asuitable assay measures the production of reducing ends created by theenzyme mixture acting on CMC wherein 1 unit is the amount of enzyme thatliberates 1 μmoL of product/min (Ghose, T. K., Pure & Appl. Chem. 1987,59, pp. 257-268).

The whole cellulase can be a β-glucosidase-enriched cellulase. Theβ-glucosidase-enriched whole cellulase generally comprises aβ-glucosidase and a whole cellulase preparation. Theβ-glucosidase-enriched whole cellulase compositions can be produced byrecombinant means. For example, such a whole cellulase preparation canbe achieved by expressing a β-glucosidase in a microorganism capable ofproducing a whole cellulase. The β-glucosidase-enriched whole cellulasecomposition can also, for example, comprise a whole cellulasepreparation and a β-glucosidase. For instance, theβ-glucosidase-enriched whole cellulase composition can suitably compriseat least 5 wt. %, 7 wt. %, 10 wt. %, 15 wt. % or 20 wt. %, and up to 25wt. %, 30 wt. %, 35 wt. %, 40 wt. %, or 50 wt. % β-glucosidase based onthe total weight of proteins in that blend/composition.

6.3.6 Xvlanases

The enzyme blends/compositions of the disclosure, for example, can,comprise one or more Group A xylanases, which may be a Trichodermareesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5.Suitable Trichoderma reesei Xyn2, Trichoderma reesei Xyn3, AfuXyn2, orAfuXyn5 polypeptides are described in Section 6.1 above.

The enzyme blends/compositions of the disclosure optionally comprise oneor more xylanases in addition to or in place of the one or more Group Axylanases. Any xylanase (EC 3.2.1.8) can be used as the additional oneor more xylanases. Suitable xylanases include, e.g., a Caldocellumsaccharolyticum xylanase (Luthi et al. 1990, Appl. Environ. Microbiol.56(9):2677-2683), a Thermatoga maritima xylanase (Wnterhalter & Liebel,1995, Appl. Environ. Microbiol. 61(5):1810-1815), a Thermatoga Sp.Strain FJSS-B.1 xylanase (Simpson et al. 1991, Biochem. J. 277,413-417), a Bacillus circulans xylanase (BcX) (U.S. Pat. No. 5,405,769),an Aspergillus niger xylanase (Kinoshita et al. 1995, Journal ofFermentation and Bioengineering 79(5):422-428), a Streptomyces lividansxylanase (Shareck et al. 1991, Gene 107:75-82; Morosoli et al. 1986Biochem. J. 239:587-592; Kluepfel et al. 1990, Biochem. J. 287:45-50), aBacillus subtilis xylanase (Bernier et al. 1983, Gene 26(1):59-65), aCellulomonas fimi xylanase (Clarke et al., 1996, FEMS MicrobiologyLetters 139:27-35), a Pseudomonas fluorescens xylanase (Gilbert et al.1988, Journal of General Microbiology 134:3239-3247), a Clostridiumthermocellum xylanase (Dominguez et al., 1995, Nature Structural Biology2:569-576), a Bacillus pumilus xylanase (Nuyens et al. AppliedMicrobiology and Biotechnology 2001, 56:431-434; Yang et al. 1998,Nucleic Acids Res. 16(14B):7187), a Clostridium acetobutylicum P262xylanase (Zappe et al. 1990, Nucleic Acids Res. 18(8):2179), or aTrichoderma harzianum xylanase (Rose et al. 1987, J. Mol.Bio1.194(4):755-756).

The xylanase can be produced by expressing an endogenous or exogenousgene encoding a xylanase. The xylanase can be, in some circumstances,overexpressed or underexpressed.

6.3.7 β-Xylosidases

The enzyme blends/compositions of the disclosure, for example, cansuitablycomprise one or more β-xylosidases. For example, theβ-xylosidase is a Group 1 β-xylosidase enzyme (e.g., an Fv3A or anFv43A) or a Group 2 β-xylosidase enzyme (e.g., a Pf43A, an Fv43D, anFv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, or a Trichodermareesei Bxl1). These polypeptides are described in Section 0 above. Forexample, an enzyme blend/composition of the disclosure can suitablycomprise one or more Group 1 β-xylosidases and one or more Group 2β-xylosidases.

The enzyme blends/compositions of the disclosure can optionally compriseone or more β-xylosidases, in addition to or in place of the Group 1and/or Group 2 β-xylosidases above. Any β-xylosidase (EC 3.2.1.37) canbe used as the additional β-xylosidases. Suitable β-xylosidases include,for example, a Talaromyces emersonii Bxl1 (Reen et al. 2003, BiochemBiophys Res Commun. 305(3):579-85), a Geobacillus stearothermophilusβ-xylosidases (Shallom et al. 2005, Biochemistry 44:387-397), aScytalidium thermophilum β-xylosidases (Zanoelo et al. 2004, J. Ind.Microbiol. Biotechnol. 31:170-176), a Trichoderma lignorum β-xylosidases(Schmidt, 1998, Methods Enzymol. 160:662-671), an Aspergillus awamoriβ-xylosidases (Kurakake et al. 2005, Biochim. Biophys. Acta1726:272-279), an Aspergillus versicolor β-xylosidases (Andrade et al.2004, Process Biochem. 39:1931-1938), a Streptomyces sp. β-xylosidases(Pinphanichakarn et al. 2004, World J. Microbiol. Biotechnol.20:727-733), a Thermotoga maritima β-xylosidases (Xue and Shao, 2004,Biotechnol. Lett. 26:1511-1515), a Trichoderma sp. SY β-xylosidases (Kimet al. 2004, J. Microbiol. Biotechnol. 14:643-645), an Aspergillus nigerβ-xylosidases (Oguntimein and Reilly, 1980, Biotechnol. Bioeng.22:1143-1154), or a Penicillium wortmanni β-xylosidases (Matsuo et al.1987, Agric. Biol. Chem. 51:2367-2379).

The β-xylosidase can be produced by expressing an endogenous orexogenous gene encoding a β-xylosidase. The β-xylosidase can be, in somecircumstances, overexpressed or underexpressed.

6.3.8 L-α-Arabinofuranosidases

The enzyme blends/compositions of the disclosure can, for example,suitably comprise one or more L-α-arabinofuranosidases. TheL-α-arabinofuranosidase is, for example, an Af43A, an Fv43B, a Pf51A, aPa51A, or an Fv51A. Af43A, Fv43B, Pf51A, Pa51A, and Fv51A polypeptidesare described in Section 6.1 above.

The enzyme blends/compositions of the disclosure optionally comprise oneor more L-α-arabinofuranosidases in addition to or in place of theforegoing L-α-arabinofuranosidases. L-α-arabinofuranosidases (EC3.2.1.55) from any suitable organism can be used as the additionalL-α-arabinofuranosidases. Suitable L-α-arabinofuranosidases include,e.g., an L-α-arabinofuranosidases of Aspergillus oryzae (Numan & Bhosle,J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Aspergillus sojae(Oshima et al. J. Appl. Glycosci. 2005, 52:261-265), Bacillus brevis(Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260),Bacillus stearothermophilus (Kim et al., J. Microbiol. Biotechnol.2004,14:474-482), Bifidobacterium breve (Shin et al., Appl. Environ.Microbiol. 2003, 69:7116-7123), Bifidobacterium longum (Margolles etal., Appl. Environ. Microbiol. 2003, 69:5096-5103), Clostridiumthermocellum (Taylor et al., Biochem. J. 2006, 395:31-37), Fusariumoxysporum (Panagiotou et al., Can. J. Microbiol. 2003, 49:639-644),Fusarium oxysporum f. sp. dianthi (Numan & Bhosle, J. Ind. Microbiol.Biotechnol. 2006, 33:247-260), Geobacillus stearothermophilus T-6(Shallom et al., J. Biol. Chem. 2002, 277:43667-43673), Hordeum vulgare(Lee et al., J. Biol. Chem. 2003, 278:5377-5387), Penicilliumchrysogenum (Sakamoto et al., Biophys. Acta 2003, 1621:204-210),Penicillium sp. (Rahman et al., Can. J. Microbiol. 2003, 49:58-64),Pseudomonas cellulosa (Numan & Bhosle, J. Ind. Microbiol. Biotechnol.2006, 33:247-260), Rhizomucor pusillus (Rahman et al., Carbohydr. Res.2003, 338:1469-1476), Streptomyces chartreusis, Streptomycesthermoviolacus, Thermoanaerobacter ethanolicus, Thermobacillusxylanilyticus (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006,33:247-260), Thermomonospora fusca (Tuncer and Ball, Folia Microbiol.2003, (Praha) 48:168-172), Thermotoga maritima (Miyazaki, Extremophiles2005, 9:399-406), Trichoderma sp. SY (Jung et al. Agric. Chem.Biotechnol. 2005, 48:7-10), Aspergillus kawachii (Koseki et al.,Biochim. Biophys. Acta 2006, 1760:1458-1464), Fusarium oxysporum f. sp.dianthi (Chacon-Martinez et al., Physiol.Mol. Plant Pathol.2004,64:201-208), Thermobacillus xylanilyticus (Debeche et al., ProteinEng. 2002, 15:21-28), Humicola insolens, Meripilus giganteus (Sorensenet al., Biotechnol. Prog. 2007, 23:100-107), or Raphanus sativus (Kotakeet al. J. Exp. Bot. 2006, 57:2353-2362).

The L-α-arabinofuranosidase can be produced by expressing an endogenousor exogenous gene encoding an L-α-arabinofuranosidase. TheL-α-arabinofuranosidase can be, in some circumstances, overexpressed orunderexpressed.

6.3.9 Accessory Proteins

The enzyme blends/compositions of the disclosure can, for example,suitably further comprise one or more accessory proteins. Examples ofaccessory proteins include, without limitation, mannanases (e.g.,endomannanases, exomannanases, and β-mannosidases), galactanases (e.g.,endo- and exo-galactanases), arabinases (e.g., endo-arabinases andexo-arabinases), ligninases, amylases, glucuronidases, proteases,esterases (e.g., ferulic acid esterases, acetyl xylan esterases,coumaric acid esterases or pectin methyl esterases), lipases, glycosidehydrolase Family 61 polypeptides, xyloglucanases, CIP1, CIP2, swollenin,expansins, and cellulose disrupting proteins. Examples of accessoryproteins can also include CIP1-like proteins, CIP2-like proteins,cellobiose dehydrogenases and manganese peroxidases. In particularembodiments, the cellulose disrupting proteins are cellulose bindingmodules.

6.4 Further Applications

In addition to saccharification of biomass, the enzymes and/or enzymeblends/compositions of the disclosure can be used in industrial,agricultural, food and feed, as well as food and feed supplementprocessing processes. Exemplary applications are described below.

6.4.1 Wood, Paper and Pulp Treatments

The enzymes, enzyme blends/compositions, and methods of the disclosurecan be used in wood, wood product, wood waste or by-product, paper,paper product, paper or wood pulp, Kraft pulp, or wood or paperrecycling treatment or industrial process. These processes include,e.g., treatments of wood, wood pulp, paper waste, paper, or pulp, ordeinking of wood or paper. The enzymes, enzyme blends/compositions ofthe disclosure can be, for example, used to treat/pretreat paper pulp,or recycled paper or paper pulp, and the like. The enzymes, enzymeblends/compositions of the disclosure can be used to increase the“brightness” of the paper when they are included in the paper, pulp,recycled paper or paper pulp treatment/pretreatment. It can beappreciated that the higher the grade of paper, the greater thebrightness; the brightness can impact the scan capability of opticalscanning equipment. As such, the enzymes, enzyme blends/compositions,and methods/processes can be used to make high grade, “bright” papers,including inkjet, laser and photo printing quality paper.

The enzymes, enzyme blends/compositions of the disclosure can be used toprocess or treat a number of other cellulosic material, including, e.g.,fibers from wood, cotton, hemp, flax or linen.

Accordingly, the disclosure provides wood, wood pulp, paper, paper pulp,paper waste or wood or paper recycling treatment processes using anenzyme, enzyme blend/composition of the disclosure.

The enzymes, enzyme blends/compositions of the disclosure can be usedfor deinking printed wastepaper, such as newspaper, or for deinkingnoncontact-printed wastepaper, e.g., xerographic and laser-printedpaper, and mixtures of contact and noncontact-printed wastepaper, asdescribed in U.S. Pat. Nos. 6,767,728 or 6,426,200; Neo, J. Wood Chem.Tech. 1986, 6(2):147. They can also be used to produce xylose from apaper-grade hardwood pulp in a process involving extracting xylancontained in pulp into a liquid phase, subjecting the xylan contained inthe obtained liquid phase to conditions sufficient to hydrolyze xylan toxylose, and recovering the xylose. The extracting step, for example, caninclude at least one treatment of an aqueous suspension of pulp or analkali-soluble material by an enzyme or an enzyme blend/composition(see, U.S. Pat. No. 6,512,110). The enzymes, enzyme blends/compositionsof the disclosure can be used to dissolve pulp from cellulosic fiberssuch as recycled paper products made from hardwood fiber, a mixture ofhardwood fiber and softwood fiber, waste paper, e.g., from unprintedenvelopes, de-inked envelopes, unprinted ledger paper, de-inked ledgerpaper, and the like, as described in, e.g., U.S. Pat. No. 6,254,722.

6.4.2 Treating Fibers and Textiles

The disclosure provides methods of treating fibers and fabrics using oneor more enzymes, enzyme blends/compositions of the disclosure. Theenzymes, enzyme blends/compositions can be used in any fiber- orfabric-treating method, which are known in the art. See, e.g., U.S. Pat.Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751; 5,980,581;U.S. Patent Publication No. 20020142438 A1. For example, enzymes, enzymeblends/compositions of the disclosure can be used in fiber and/or fabricdesizing. The feel and appearance of a fabric can be, for example,improved by a method comprising contacting the fabric with an enzyme orenzyme blend/composition of the disclosure in a solution. Optionally,the fabric is treated with the solution under pressure. The enzymes,enzyme blends/composition of the disclosure can also be used to removestains.

The enzymes, enzyme blends/compositions of the disclosure can be used totreat a number of other cellulosic material, including fibers (e.g.,fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g.,knits, wovens, denims, yarns, and toweling, made from cotton, cottonblends or natural or manmade cellulosics or blends thereof. The textiletreating processes can be used in conjunction with other textiletreatments, e.g., scouring and/or bleaching. Scouring, for example, isthe removal of non-cellulosic material from the cotton fiber, e.g., thecuticle (mainly consisting of waxes) and primary cell wall (mainlyconsisting of pectin, protein and xyloglucan).

6.4.3 Treating Foods and Food Processing

The enzymes, enzyme blends/compositions of the disclosure have numerousapplications in food processing industry. They can, for example, be usedto improve extraction of oil from oil-rich plant material, e.g.,oil-rich seeds. The enzymes, enzyme blends/compositions of thedisclosure can be used to extract soybean oil from soybeans, olive oilfrom olives, rapeseed oil from rapeseed, or sunflower oil from sunflowerseeds.

The enzymes, enzyme blends/compositions of the disclosure can also beused to separate components of plant cell materials. For example, theycan be used to separate plant cells into components. The enzymes, enzymeblends/ compositions of the disclosure can also be used to separatecrops into protein, oil, and hull fractions. The separation process canbe performed using known methods.

The enzymes, enzyme blends/compositions of the disclosure can, inaddition to the uses above, be used to increase yield in the preparationof fruit or vegetable juices, syrups, extracts and the like. They canalso be used in the enzymatic treatment of various plant cellwall-derived materials or waste materials from, e.g., cereals, grains,wine or juice production, or agricultural residues such as, e.g.,vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp,and the like. Further, they can be used to modify the consistency and/orappearance of processed fruits or vegetables. Additionally, they can beused to treat plant material so as to facilitate processing of the plantmaterial (including foods), purification or extraction of plantcomponents. The enzymes, enzyme blends/compositions of the disclosurecan be used to improve feed value, decrease the water binding capacity,improve the degradability in waste water plants and/or improve theconversion of plant material to ensilage, and the like.

The enzymes, enzyme blends/compositions of the disclosure can also beused in baking applications. In some embodiments, they are used tocreate non-sticky doughs that are not difficult to machines and toreduce biscuit sizes. They can also be used to hydrolyze arabinoxylansto prevent rapid rehydration of the baked product that can lead to lossof crispiness and reduced shelf-life. For example, they are used asadditives in dough processing. 6.4.4 Animal Feeds and Food or Feed orFood Additives

The disclosure provides methods for treating animal feeds and foods andfood or feed additives (supplements) using enzymes, enzymeblends/compositions of the disclosure. Animals including mammals (e.g.,humans), birds, fish, and the like. The disclosure provides animalfeeds, foods, and additives (supplements) comprising enzymes, enzymeblends/compositions of the disclosure. Treating animal feeds, foods andadditives using enzymes of the disclosure can help in the availabilityof nutrients, e.g., starch, protein, and the like, in the animal feed oradditive (supplements). By breaking down difficult to digest proteins orindirectly or directly unmasking starch (or other nutrients), theenzymes, enzyme blends/compositions can make nutrients more accessibleto other endogenous or exogenous enzymes. They can also simply cause therelease of readily digestible and easily absorbed nutrients and sugars.

When added to animal feed, enzymes, enzyme blends/compositions of thedisclosure improve the in vivo break-down of plant cell wall materialpartly by reducing the intestinal viscosity (see, e.g., Bedford et al.,Proceedings of the 1st Symposium on Enzymes in Animal Nutrition, 1993,pp. 73-77), whereby a better utilization of the plant nutrients by theanimal is achieved. Thus, by using enzymes, enzyme blends/compositionsof the disclosure in feeds, the growth rate and/or feed conversion ratio(i.e., the weight of ingested feed relative to weight gain) of theanimal can be improved.

The animal feed additive of the disclosure may be a granulated enzymeproduct which can be readily mixed with feed components. Alternatively,feed additives of the disclosure can form a component of a pre-mix. Thegranulated enzyme product of the disclosure may be coated or uncoated.The particle size of the enzyme granulates can be compatible with thatof the feed and/or the pre-mix components. This provides a safe andconvenient mean of incorporating enzymes into feeds. Alternatively, theanimal feed additive of the disclosure can be a stabilized liquidcomposition. This may be an aqueous- or oil-based slurry. See, e.g.,U.S. Pat. No. 6,245,546.

An enzyme, enzyme blend/composition of the disclosure can be supplied byexpressing the enzymes directly in transgenic feed crops (e.g., astransgenic plants, seeds and the like), such as grains, cereals, corn,soy bean, rape seed, lupin and the like. As discussed above, thedisclosure provides transgenic plants, plant parts and plant cellscomprising a nucleic acid sequence encoding a polypeptide of thedisclosure. The nucleic acid is expressed such that the enzyme of thedisclosure is produced in recoverable quantities. The xylanase can berecovered from any plant or plant part. Alternatively, the plant orplant part containing the recombinant polypeptide can be used as suchfor improving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The disclosure provides methods for removing oligosaccharides from feedprior to consumption by an animal subject using an enzyme, enzymeblend/composition of the disclosure. In this process a feed is formed tohave an increased metabolizable energy value. In addition to enzymes,enzyme blends/compositions of the disclosure, galactosidases,cellulases, and combinations thereof can be used.

The disclosure provides methods for utilizing an enzyme, an enzymeblend/composition of the disclosure as a nutritional supplement in thediets of animals by preparing a nutritional supplement containing arecombinant enzyme of the disclosure, and administering the nutritionalsupplement to an animal to increase the utilization of hemicellulasecontained in food ingested by the animal.

6.4.5 Waste Treatment

The enzymes, enzyme blends/compositions of the disclosure can be used ina variety of other industrial applications, e.g., in waste treatment.For example, in one aspect, the disclosure provides a solid wastedigestion process using the enzymes, enzyme blends/compositions of thedisclosure. The methods can comprise reducing the mass and volume ofsubstantially untreated solid waste. Solid waste can be treated with anenzymatic digestive process in the presence of an enzymatic solution(including the enzymes, enzyme blends/compositions of the disclosure) ata controlled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See, e.g., U.S. Pat. No. 5,709,796.

6.4.6 Detergent, Disinfectant and Cleaning Compositions

The disclosure provides detergent, disinfectant or cleanser (cleaning orcleansing) compositions comprising one or more enzymes, enzymeblends/compositions of the disclosure, and methods of making and usingthese compositions. The disclosure incorporates all known methods ofmaking and using detergent, disinfectant or cleanser compositions. See,e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561; 6,380,147.

In specific embodiments, the detergent, disinfectant or cleansercompositions can be a one- and two-part aqueous composition, anon-aqueous liquid composition, a cast solid, a granular form, aparticulate form, a compressed tablet, a gel and/or a paste and a slurryform. The enzymes, enzyme blends/compositions of the disclosure can alsobe used as a detergent, disinfectant, or cleanser additive product in asolid or a liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions, and can be added at any stage of the cleaning process.

The present disclosure provides cleaning compositions includingdetergent compositions for cleaning hard surfaces, detergentcompositions for cleaning fabrics, dishwashing compositions, oralcleaning compositions, denture cleaning compositions, and contact lenscleaning solutions.

When the enzymes of the disclosure are components of compositionssuitable for use in a laundry machine washing method, the compositionscan comprise, in addition to an enzyme, enzyme blend/composition of thedisclosure, both a surfactant and a builder compound. They canadditionally comprise one or more detergent components, e.g., organicpolymeric compounds, bleaching agents, additional enzymes, sudssuppressors, dispersants, lime-soap dispersants, soil suspension andanti-redeposition agents, and corrosion inhibitors.

Laundry compositions of the disclosure can also contain softeningagents, as additional detergent components. Such compositions containingcarbohydrase can provide fabric cleaning, stain removal, whitenessmaintenance, softening, color appearance, dye transfer inhibition andsanitization when formulated as laundry detergent compositions.

The disclosure thus further provides a process of saccharification abiomass material comprising hemicellulose. Such a biomass material canoptionally comprise cellulose. Exemplary biomass materials include,without limitation, corcob, switchgrass, sorghum, and/or bagasse.Accordingly the disclosure provides a process of saccharification,comprising treating a biomass material herein comprising hemicelluoseand optionally cellose with an enzyme blend/composition as describedherein. The enzyme blend/compositon used in such a processs of theinvention include 0.5 g to 40 g (e.g., 0.5 g to 20 g, 0.5 g to 30 g, 0.5g to 40 g, 0.5 g to 15 g, 0.5 g to 10 g, 0.5 g to 5 g, 0.5 g to 7 g,etc) of polypeptides having xylanase activity per kg of hemicellulose inthe biomass material. The enzyme blend/composition used in such aprocess of the invention can also include 1 g to 40 g (e.g., 2 g to 20g, 3 g to 7 g, 1 g to 5 g, or 2 g to 5 g, etc.) of polypeptides havingxylanase activity per kg of hemicellulose in the biomass material. Theenzyme blend/composition used in such a process can include 0.5 g to 50g (e.g., 0.5 g to 50 g, 0.5 g to 45 g, 0.5 g to 40 g, 0.5 g to 30 g, 0.5g to 25 g, 0.5 g to 20 g, 0.5 g to 15 g, 0.5 g to 10 g, etc) ofpolypeptides having β-xylosidase activity per kg of hemicellulose in thebiomass material. The enzyme blend/composition used in such a processcan also include 1 g to 50 g (e.g., 2 g to 40 g, 4 g to 20 g, 4 g to 10g, 2 g to 10 g, 3 g to 7 g, etc.) of polypeptide having β-xylosidaseactivity per kg of hemicellulose in the biomass material. The enzymeblend/compositon used in such a process of the invention can include 0.2g to 20 g (e.g., 0.2 g to 18 g, 0.2 g to 15 g, 0.3 g to 10 g, 0.2 g to 8g, 0.2 g to 5 g, etc) of polypeptides having L-α-arabinofuranosidaseactivity per kg of hemicellolose in the biomass material. The enzymeblend/composition used in such a process of the invention can include0.5 g to 20 g (e.g., 1 g to 10 g, 1 g to 5 g, 2 g to 6 g, 0.5 g to 4 g,or 1 g to 3 g, etc) of polypeptides having L-α-arabinofuranosidaseactivity per kg of hemicellolose in the biomass material. The enzymeblend/composition can also include 1 g to 100 g (e.g., 1 g to 100 g, 2 gto 80 g, 3 g to 50 g, 5 g to 40 g, 2 g to 20 g, 10 g to 30 g, or 12 g to18 g, etc) of polypeptides having cellulase activity per kg of cellulosein the biomass material. Optionally, the amount of polypeptides havingβ-glucosidase activity can constitute up to 50% of the total weight ofpolypeptides having cellulase activity.

A suitable process of the invention preferably yields 60% to 90% xylosefrom the hemicellulose xylan of the biomass material treated. Suitablebiomass materials include one or more of, for example, corncob,switchgrass, sorghum, and/or bagasse. As such, a process of theinvention preferably yields at least 70% (e.g. at least 75%, at least80%) xylose from hemicellulose xylan from one or more of these biomassmaterials. For example, the process yields 60% to 90% of xylose fromhemicellulose xylan of a biomass material comprising hemicellulose,including, without limitation, corncob, switchgrass, sorghum, and/orbagasse.

The process of the invention optionally further comprises recoveringmonosaccharides.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

7. EXAMPLE 1: REPRESENTATIVE EXPERIMENTAL METHODS

7.1 Materials and Methods

The following assays/methods were used in Example 1 and subsequentexamples. Any deviations from the protocols provided below areindicated.

7.1.1 Preparation of Hemicellulose from Plant Tissues

Hemicellulose preparations were prepared using a modification of theNaOH/sonication procedure described by Erbringerova et al. (CarbohydratePolymers 1998, 37:231). Dry plant material was ground to pass a 1 mmscreen and 10 g of this material was suspended in 250 mL of 5% (wt/v)NaOH. The suspension was heated to 80° C. without stirring for 30 minthen sonicated for 15 min at ambient temperature using a probe sonicatorat high setting. The suspension was returned to 80° C. for an additional30 min then allowed to cool to room temperature. Solids were removedfrom the suspension by centrifugation at 3000×g for 15 min and theresulting supernatant was decanted into 1 L of ethanol which was cooledon ice. After 30 min the resulting precipitate was recovered by firstdecanting the clear liquid above the precipitate then filtration withoutallowing the precipitate to fully air dry. The filter cake was firstwashed with 200 mL of cold, 80% ethanol then removed from the filterwithout allowing it to air dry. The filter cake was re-dissolved in 200mL of water and the pH of the solution was adjusted to 5.5 with aceticacid. The extracted carbohydrate was re-precipitated by addition to 1 Lof ethanol on ice and the resulting precipitate was again recovered byfiltration as above. The filter cake was frozen and remaining solventand water was removed by lyophylization. Yield of recovered carbohydrateranged from 6 to 23% of the starting plant material depending on thetissue and the preparation.

7.1.2 Dilute Ammonia Pretreatment of Biomass Substrates

Corncob and switchgrass were pretreated prior to enzymatic hydrolysisaccording to the methods and processing ranges described in WO06110901A(unless otherwise noted).

7.1.3 Compositional Analysis of Biomass

The 2-step acid hydrolysis methods described in “Determination ofstructural carbohydrates and lignin in the biomass” (National RenewableEnergy Laboratory, Golden, Colo. 2008, available atwww.nrel.gov/biomass/pdfs/42618.pdf) were used to measure thecomposition of biomass substrates. Enzymatic hydrolysis results arereported in terms of percent conversion with respect to the theoreticalyield from the starting glucan and xylan content of the substrate.

7.1.4 Preparation of Crude Oligomers from Ammonia Pretreated Corncob

Crude oligomers for screening hemicellulases were prepared from corncobby the following procedure. Hammer-milled corncob (˜1/4 in meandiameter) plus 6% ammonia (w/w) was heated to 145° C. with directinjection of steam into a stirred pressure reactor. After 20 min excessammonia was flashed out of the reactor at a final vacuum of ˜0.1 bar.The ammonia pretreated cob was then placed in a sterile stirred reactorfor enzyme saccharification. Enough water was added to obtain a finaltotal solids loading of 25% (w/w) after all additions are made. The pHof the reactor was maintained at pH 5.3 with 4 N sulfuric acid and thetemperature controlled at 47° C. Spezyme® CP, Multifect® Xylanase(Danisco US Inc., Genencor), and Novo 188 (Novozymes, Denmark) wereadded at loadings of 20, 10 and 5 mg/g of cellulose, respectively, andallowed to saccharify the pretreated cob to sugars and oligomers for 116h. The material was then cooled to 33° C. and the pH adjusted to 5.8with 4 N NaOH. The glucose and xylose were then fermented to ethanol byadding a seed culture of a recombinant Zymomonas mobilis strain (10%total volume, ATCC accession no. PTA-1798) as described in U.S. Pat. No.7,354,755. The fermentation progress was followed until all of theglucose and ˜95% of the xylose was consumed. A 0.5 L aliquot of thefermentation broth was clarified by centrifugation (21,000×g) for 20 minfollowed by filtration of the supernatant through a 0.2 micron filteringunit (Nalgene). The ethanol was removed from the filtered fermentationbroth on a rotovap maintained at 35° C. under house vacuum. The totalvolume of the final liquor was reduced by ˜4× by the latter procedure.

7.1.5 Total Protein Assays

Different total protein determination methods were employed depending onthe nature of the protein sample (i.e., purified, fermentation broth,commercial product, etc.). The BCA protein assay is an example of acolorimetric assay that measures protein concentration with aspectrophotometer.

Reagents: BCA Protein Assay Kit (Pierce Chemical, Product #23227), 50 mMSodium Acetate buffer pH 5.0, 15% trichloroacetic acid (TCA), 0.1 NNaOH, BSA stock solution, Reagent A, Reagent B (from protein assay kit)

Procedure: Enzyme dilutions were prepared in test tubes using 50 mMSodium Acetate buffer. Diluted enzyme solution (0.1 mL) was added to 2mL Eppendorf centrifuge tubes containing 1 mL 15% TCA. The tubes werevortexed and placed in an ice bath for 10 min. The samples were thencentrifuged at 14,000 rpm for 6 min. The supernatant was poured out, thepellet resuspended in 1 mL 0.1 N NaOH, and the tubes vortexed until thepellet dissolved. BSA standard solutions were prepared from a stocksolution of 2 mg/mL. BCA working solution was prepared by mixing 0.5 mLReagent B with 25 mL Reagent A. The resuspended protein (0.1 mL each)was added to 3 Eppendorf centrifuge tubes. Two mL Pierce BCA workingsolution was added to each of the sample and serially diluted BSAstandard Eppendorf tubes. All tubes were incubated in a 37° C. waterbath for 30 min. The samples were then cooled to room temperature (15min) and the absorbance measured at 562 nm in a spectrophotometer.

Calculations: Average values for each BSA protein standard absorbancewere calculated and plotted, absorbance on x-axis and concentration(mg/mL) on the y-axis. A linear curve fit was applied and the equationfor the line calculated using the formula: y=mx+b

The raw concentration of the enzyme samples was calculated bysubstituting the absorbance for the x-value. The total proteinconcentration was calculated by multiplying with the dilution factor.

The total protein of purified samples was determined by A280 (see, e.g.,Pace et al., Protein Science, 1995, 4:2411).

Some protein samples were measured using the Biuret method as modifiedby Weichselbaum and Gornall using Bovine Serum Albumin as a calibrator(modified Biuret) (Weichselbaum, Amer. J. Clin. Path. 1960, 16:40;Gornall et al., J. Biol. Chem. 1949, 177:752).

Total protein content of fermentation products was also sometimesmeasured as total nitrogen by combustion, capture and measurement ofreleased nitrogen, either by Kjeldahl (rtech laboratories,www.rtechlabs.com) or in-house by the DUMAS method (TruSpec CN,www.leco.com) (SADER, et al. Archives of Veterinary Science, 9(2):73-79,2004). For complex protein-containing samples, e.g. fermentation broths,an average 16% N content, and the conversion factor of 6.25 for nitrogento protein was used. In some cases, total precipitable protein wasmeasured to remove interfering non-protein nitrogen. A 12.5% final TCAconcentration was used and the protein-containing TCA pellet wasresuspended in 0.1 M NaOH.

In other cases, Coomassie Plus- the Better Bradford Assay (ThermoScientific, Rockford, Ill. product #23238) was used according tomanufacturer recommendation.

7.1.6 Synthetic Substrate (para-nitrophenyl Substrate) Activity Assays

Active protein from T. reesei expression of cloned genes was confirmedwith model substrate assays. Cellulase and hemicellulase activities onthe synthetic substrates, such as 4-nitrophenyl a-L-arabinofuranoside(pNPA, Sigma N3641) and 4-nitrophenyl β-D-glucopyranoside (pNPG, SigmaN7006), and 4-nitrophenyl β-D-xylopyranoside (pNPX, Sigma N2132) weremeasured as follows: Substrate solution was prepared by dissolving 30 mgof synthetic substrate in 100 mL 50 mM Sodium Acetate buffer, pH 4.8.Sodium carbonate (1 M) was prepared for reaction quenching. Substratesolution (100 μL) was dispensed into Costar 96 well plates (Cat no.9017). 20 μL of enzyme sample was dispensed into a microtiter platewell. The microtiter plate was incubated at 50° C. for 10 min using aThermomixer R heating and cooling shaker (Eppendorf). 50 μL of 1 Msodium carbonate was added to each well to quench the reaction.Absorbance at 400 nm wavelength was read with SpectraMax 340C384Microplate Spectrophotometer (Molecular Devices). Units per mL weredetermined by using a p-nitrophenol standard curve. A Quad-deleteTrichoderma host, from which the cbh1, cbh2, egl1 and egl2 genes weredeleted (see WO 05/001036), was analyzed with the enzyme samples as acontrol for the activity of enzymes expressed in this background. 7.1.7Cob Saccharification Assay

Typically, Corncob saccharification in a microtiter plate format wasperformed in accordance with the following procedures. The biomasssubstrate, dilute ammonia pretreated corncob, was diluted in water andpH-adjusted with sulfuric acid to create a pH 5, 7% cellulose slurrythat was used directly in the assay. The enzymes tested included:commercial cellulase products, e.g. Accellerase® 1000, Accellerase® 1500(Danisco US Inc., Genencor), T. reesei fermentation broths, and purifiedenzymes. The enzymes were loaded based on mg total protein per gram ofcellulose (as determined by compositional analysis) in the corncobsubstrate. The enzymes were diluted in 50 mM Sodium Acetate pH 5.0 toobtain the desired loading concentration at the required volume. Fortymicroliters of enzyme solution was added to 70 mg of dilute-ammoniapretreated corncob at 7% cellulose per well (equivalent to 4.5%cellulose final). The assay plate was incubated at room temperature for10 min. The assay plates were covered with aluminum plate sealers andthe plates incubated at 50° C., 200 rpm, for three days. At the end ofthe incubation period, the saccharification reaction was quenched byadding 100 μL of 100 mM glycine buffer, pH10.0 per well and the platewas centrifuged for 5 min at 3,000 rpm. Ten microliters of thesupernatant were added to 200 μL of MilliQ water in a 96-well HPLC plateand the soluble sugars were measured by HPLC.

This describes a typical method that was used in multiple Examplesherein. In certain Examples, corncob saccharification was measured usinga modified protocol. The modifications are described with the individualexamples.

7.1.8 Suqar Analysis by HPLC

Typically, samples from cob saccharification hydrolysis were prepared bycentrifugation to clear insoluble material, filtration through a 0.22 μmnylon filter (Spin-X centrifuge tube filter, Corning Incorporated,Corning, N.Y.) and dilution to an appropriate concentration of solublesugars with distilled water. Monomer sugars were determined on a ShodexSugar SH-G SH1011, 8×300 mm with a 6×50 mm SH-1011P guard column(www.shodex.net). Solvent was 0.01 N H₂SO₄ run at 0.6 mL/min. Columntemperature was 50° C. and detection was made using a refractive indexdetector. External standards of glucose, xylose and arabinose were runwith each sample set. Certain examples herein use a protocol to achievethe same end with a somewhat modified set of protocols. The specificmodifications to the protocols are described with individual examples.

Oligomeric sugars were separated by size exclusion chromatography usinga Tosoh Biosep G2000PW column 7.5 mm×60 cm (www.tosohbioscience.de). Thesolvent was distilled water at 0.6 mL/min and the column was run at roomtemperature. Six carbon sugar standards used for size calibration were:stachyose, raffinose, cellobiose and glucose; and 5 carbon sugars were:xylohexose, xylopentose, xylotetrose, xylotriose, xylobiose and xylose.Xylo-oligomers were obtained from Megazyme (www.megazyme.com). Detectionwas by refractive index and when reported quantitatively results areeither as peak area units or relative peak areas by percent.

Total soluble sugars were determined by acid hydrolysis of thecentrifuged and filter clarified samples described above. The clarifiedsample was diluted 1:1 with 0.8 N H₂SO₄ and the resulting solution wasautoclaved in a capped vial for a total cycle time of 1 h at 121° C.Results are reported without correction for loss of monomer sugar duringthe hydrolysis.

7.1.9 Protein Analysis by HPLC

To separate and quantify the enzymes contained in broth from 14Lfermentations of the integrated expression strains, liquidchromatography (LC) and mass spectroscopy (MS) were performed. Enzymesamples were first treated with a recombinantly expressed endoHglycosidase from S. plicatus (e.g., NEB P0702L). EndoH was used at aratio of 0.01-0.03 μg endoH protein per pg sample total protein andincubated for 3 h at 37° C., pH 4.5-6.0 to enzymatically remove N-linkedgycosylation prior to HPLC analysis. Approximately 50 μg of protein wasthen injected for hydrophobic interaction chromatography using anAgilent 1100 HPLC system with an HIC-phenyl column and a high-to-lowsalt gradient over 35 min was performed on samples of concentratedfermentation broth. The gradient was achieved using high salt buffer A:4 M ammonium sulphate containing 20 mM potassium phosphate pH 6.75 andlow salt buffer B: 20 mM potassium phosphate pH 6.75. Peaks weredetected with UV light at 222 nm and fractions were collected andidentified by mass spectroscopy.

7.1.10 Cellulase Activity Assay Using Calcofluor White

Cellulase activity was measured on PASO using a calcofluor whitedetection method (Appl. Biochem. Biotechnol. 161:313-317). All chemicalsused were of analytical grade. Avicel PH-101 was purchased from FMCBioPolymer (Philadelphia, Pa.). Calcofluor white was purchased fromSigma (St. Louis, Mo.). Phosphoric acid swollen cellulose (PASO) wasprepared from Avicel PH-101 using an adapted protocol of Walseth, TAPPI1971, 35:228 and Wood, Biochem. J. 1971, 121:353-362. In short, Avicelwas solubilized in concentrated phosphoric acid then precipitated usingcold deionized water. After the cellulose was collected and washed withmore water to neutralize the pH, it was diluted to 1% solids in 50 mMSodium Acetate buffer, pH 5.0.

All enzyme dilutions were made into 50 mM Sodium Acetate buffer, pH 5.0.GC220 Cellulase (Danisco US Inc., Genencor) was diluted to 2.5, 5, 10,and 15 mg protein/g PASO, to produce a linear calibration curve. Samplesto be tested were diluted to fall within the range of the calibrationcurve, i.e. to obtain a response of 0.1 to 0.4 fraction product. 150 μLof cold 1% PASC was added to 20 μL of enzyme solution in 96-wellmicrotiter plates. The plate was covered and incubated for 2 h at 50°C., 200 rpm in an Innova incubator/shaker. The reaction was quenchedwith 100 μL of 50 μg/mL Calcofluor in 100 mM Glycine, pH 10.Fluorescence was read on a fluorescence microplate reader (SpectraMax M5by Molecular Devices) at excitation wavelength Ex=365 nm and emissionwavelength Em=435 nm. The result (shown in FIG. 25) is expressed as thefraction product according to the equation:

FP=1−(FI sample−FI buffer)/(FI zero enzyme−FI buffer),

wherein FP is fraction product, and FI=fluorescence units.

7.1.11 Cultivation of Fusarium Verticillioides and Purification ofmMajor Hemicellulase Activities Detected in the Extracellular Protein

Wild type Fusarium verticillioides source was as described in Table 1 ofFuchs et al., Fungal Genetics and Biology 2004, 41:852-863. The funguswas grown using destarched corn grain fiber using a modification of themethod described in Li et al., Applied Biochemistry and Biotechnology2005, (121-124):321-334.

Starch was removed from the dried corn pericarp fraction from a corn drymill fractionation by slurrying 200 g of corn pericarp in 3 L of tapwater and heating to 80° C. with 5 mL (500 mg) of high temperatureamylase Liquozyme SC DS (Novozymes, Denmark). The mixture was stirredoccasionally with a spatula and held at 80° C. for 30 min then allowedto cool to room temperature for about 2 h. The resulting slurry wasdecanted onto a 20 mesh screen to partially de-water. The solids on thescreen were further washed with 4 L of tap water then air driedovernight before a final drying in an oven at 60° C. The dried materialwas ground to pass a 1 mm screen in a knife mill before use.

The F. verticillioides culture was maintained on potato dextrose agar(Sigma P6685) and a rich growth media of 24 g/L potato dextrose wasinoculated with mycelia from the plate. The growth culture was incubatedfor 3 days at 30° C. with agitation at 130 rpm. After 3 days growth the150 mL of the resulting cell mass and spent media were used to inoculatea corn pericarp induction media. The induction media was 80 gde-starched corn pericarp in 1 L base media (15 g KH₂PO₄, 5 g ammoniumsulfate, 20 g yeast extract, 0.5 g magnesium sulfate and 1 g Tween 80 atpH4.8). The culture was maintained at 30° C. with agitation at 130 rpmfor 7 days.

Extracellular protein was separated from the spent grain and fungal cellmass by centrifugation at 2,000×G for 20 min. The partially clarifiedsupernatant was passed first through a 0.45 μm filter and then through a0.22 μm filter. The clarified filtrate was stored at 4° C. until used.The filtrate was concentrated approximately 14-fold with Amicon ultra-4centrifugal filters with a 30 kD cutoff (Millipore).

L-α-arabinofuranosidase (LARF) and β-xylosidase (BXL) activities werepurified from the fungal supernatant by first exchanging the proteinsinto 50 mM pH 6.3 (N-Morpholino) ethanesulphonic acid-NaOH (MES) buffer,then applying protein onto a GE Health Sciences Q-Sepharose Fast Flowquaternary amine anion exchange column (20 mL bed volume equilibratedwith MES buffer). The protein purification system used was a GH HealthSciences Akta system using Unicorn software. Proteins were eluted with a0-0.5 M NaCI gradient in MES buffer.

Activity in column fractions was monitored using either 4-nitrophenylβ-D-xylopyranoside (for BXL activity) or 4-nitrophenylα-L-arabinofuranoside (for LARF activity). In both cases, 2 μL of thecolumn fraction was added to 95 μL of 50 mM pH 5.0 Sodium Acetate bufferand 5 μL of the p-nitrophenyl substrate, mixed and incubated 2 min at23° C. 100 μL of 1 N sodium carbonate stop solution was added andabsorbance at 405 nm was read.

Active enzyme fractions from the anion exchange system were collected,concentrated on Amicon Centriprep filtration, and applied to a GE HealthSciences SP-Sepharose Fast Flow sulfopropyl cation exchange column (20mL bed equilibrated with Sodium Acetate buffer) using the same proteinpurification system described above. Proteins were eluted with a 0-1 MNaCI gradient in Sodium Acetate buffer, and activity in the fractionswas monitored as described above. Active fractions were then collected,pooled and concentrated if necessary. Proteins in active fractions wereidentified by MS-MS.

7.1.12 Protein Identification by MS-MS

The identification of a representative set of Fusarium verticillioidesfamily GH54 L-α-arabinofuranosidase (LARF), family GH51 LARF, two familyGH3 β-xylosidases and a family GH30 β-xylosidase (BXL) is describedbelow.

For the identification of GH54 LARF, 200 μL of 1 N HCl (EM Industries,Hawthorne, N.Y.) was added to 250 μL of “Fraction B3” (0.35 mg protein)from the purification procedure described above and the sample iced for10 min. Next, 200 μL of 50% TCA (Sigma-Aldrich, St. Louis, Mo.) wasadded and samples were iced for another 10 min to precipitate protein.Samples were centrifuged for 2 min at 16,000 rcf and the supernatantswere discarded. The pelleted protein was washed with 90% cold acetone(J. T. Baker, Phillipsburg, N.J.), then centrifuged for 1 min at 16,000rcf. The supernatant was discarded and the pellet was left to air dry.First, 30 μL of 8 M urea (MP Biomedicals, Solon, Ohio) was added to thepellet, then 4 μL of 0.2 M DTT (Sigma-Aldrich, St. Louis, Mo.). Samplewas incubated at 52° C. for 30 min and then 4 μL of 0.44 M iodoacetamide(Sigma-Aldrich, St. Louis, Mo.) was added and the solution incubated atroom temperature in the dark for 30 min. Next, 120 μL of 0.1% n-octylB-D-glucopyranoside water (Sigma-Aldrich, St. Louis, Mo.) was addedslowly, and then sample was divided into two equal parts. To half thesample, 7.5 μg Trypsin (Promega Corp., Madison, Wis.) was added, and tothe other half, 4 μg of AspN (Roche Applied Science, Indianapolis, Ind.)was added. Both samples were incubated at 37° C. for 1 h. Samples werequenched with 10% Trifluoroacetic Acid (Thermoscientific, Waltham,Mass.) for a final volume of 0.1% TFA. Both samples were run on aThermofinnigan (San Jose, Calif.) LCQ-Deca electrospray ionization (ESI)ion-trap mass spectrometer. A Vydac C18 column (5 u, 300A, 0.2×150 mm,Michrom Bioresources, Auburn, Calif.) was run at a flow rate of 200μL/min. The injection volumes were 50 μL, and were filtered through anon-line trapping cartridge (Peptide CapTrap, Michrom Bioresources,Auburn, Calif.) before loading onto the column. Separation of thedigests was performed with a gradient as follows (Solvent A: 0.1%trifluoroacetic acid in H₂O (J. T. Baker, Phillipsburg, N.J.), SolventB: 0.08% trifluoroacetic acid in acetonitrile (J. T. Baker,Phillipsburg, N.J.)). For the identification of GH51 LARF, the two GH3β-xylosidases, and GH30 β-xylosidase, the method described above wasused, except samples were digested with Chymotrypsin (7.5 μg,Sigma-Aldrich, St. Louis, Mo.) in addition to Trypsin and AspN. TheTurboSequest search engine within Bioworks 3.31 (Thermo, San Jose) wasused to identify proteins from the Fusarium verticillioides proteindatabase. The protein database was downloaded from the BROAD Instituteatwww.broadinstitute.org/annotation/genome/fusarium_verticillioides/MultiDownloads.html.

7.1.13 Induction of Fusarium verticillioides Enzymes by Dilute AqueousAmmonia Pretreated Switchgrass

For induction of Fusarium verticillioides enzymes in response to diluteaqueous ammonia pretreated switchgrass, switchgrass was ground to <1 mmdiameter, then pretreated with 6% ammonia based on dry weight at 50%initial dry matter at 160° C. maximum temperature for 90 min. Theresulting material was 43.3% solids and contained 0.336% residualammonia.

Wild type F. verticillioides culture was maintained on potato dextroseagar (Sigma P6685) and a rich growth media of 24 g/L potato dextrose wasinoculated with mycelia from the plate. The growth culture was incubatedfor 3 days at 24° C. with agitation at 140 rpm, during which time itbecame turbid with Fusarium cells. After 3 days growth, 4 flasks eachcontaining 100 mL of base Christakapoulos media (0.1% KH₂PO₄, 0.03%CaCl₂, 0.03% MgSO₄×7 H₂O, 2.61% Na₂HPO₄×7 H₂O, 0.134% NaH₂PO₄×1 H₂O,1.0% anhydrous ammonium phosphate) were inoculated with 4 mL of theresulting cell suspension. To the suspension, two grams dry matter ofdilute aqueous ammonia-pretreated switchgrass was added as the solecarbon source.

After addition of the pretreated switchgrass, the pH was adjusted to 6.5and the flask was swirled at 180 rpm at 23° C. for 168 h. Prior to sizeexclusion chromatography, samples were analyzed at different time pointsby Bradford assay for protein, p-nitrophenyl arabinofuranosidase,p-nitrophenyl xylosidase and p-nitrophenyl glucosidase activities, andby SDS-PAGE gels for induction of enzymes. Intact non-pretreatedswitchgrass, 2% glucose and no carbon source were included as parallelcontrols and were found to lead to much lower levels of enzyme inductionthan was the case for dilute aqueous ammonia-pretreated switchgrasscarbon source.

7.1.14 Size exclusion chromatography fractionation of the Fusariumverticillioides culture broth

The Fusarium verticillioides culture media containing the expressedenzymes from the 168-h induction described above was centrifuged at3,500×g to remove debris and cells. The supernatant was filtered througha 0.4 μm filter yielding a clear deep yellow liquid. The liquid wasconcentrated (˜4×) two times in a large Amicon ultra 10K MW cutoffconcentrator (UFC901024). A 1.7 mL concentrated sample was set aside forsize exclusion chromatography (SEC) and assayed for protein content byBCA assay as described earlier. The concentrated protein sample wasloaded onto a Superdex 16/60 SEC column equilibrated with 50 mM SodiumAcetate buffer pH 5.0. The column was eluted at 1 mL/min at 4° C.Absorbance of the sample at 214 nm and 280 nm were monitored. Afterelution of the void volume, 1 mL fractions were collected in a deep wellpolypropylene microtiter plate. The protein-containing fractions wereaggregated into a separate deep well plate and all fractions weremeasured for total protein concentration by BCA assay, and p-nitrophenylarabinosidase, p-nitrophenyl xylosidase, and p-nitrophenyl glucosidaseactivities as described in Example 1 (Synthetic substrate activityassays).

7.1.15 Purification of A NUMBER OF GH43, GH51 and GH3 HOMOLOGS (SECTION8.1 BELOW) from T. reesei fermentation by Cation Exchange Chromatography

Shake flask-scale enzyme production from wild type Trichoderma reeseicultures was performed as described in WO 2005/001036. The extracellularenzyme preparations were concentrated by Amicon Centriprep filtration ifnecessary, and applied to a GE Health Sciences SP-Sepharose Fast Flowsulfopropyl cation exchange column (20 mL bed equilibrated with SodiumAcetate buffer) using a GE Health Sciences Akta system proteinpurification system using Unicorn software. Proteins were eluted with a0-1 M NaCI gradient in Sodium Acetate buffer and activity in thefractions were monitored as described above. Active fractions were thencollected, pooled and concentrated if necessary. Activity in columnfractions was monitored using either 4-nitrophenyl β-D-xylopyranoside(for BXL activity) or 4-nitrophenyl α-L-arabinofuranoside (for LARFactivity). In both cases, 2 μL of the column fraction was added to 95 μLof 50 mM pH 5.0 Sodium Acetate buffer and 5 μL of the p-nitrophenylsubstrate, mixed and incubated 2 min at 23° C. One hundred microlitersof 1 N sodium carbonate stop solution was added and the absorbance wasmeasured at 405 nm. In some cases an anion exchange protein purificationstep was useful before the cation exchange. In this case, the enzymefractions were exchanged into 50 mM pH 6.3 (N-Morpholino)ethanesulphonic acid-NaOH (MES) buffer, and then the protein sample wasapplied onto a GE Health Sciences Q-Sepharose Fast Flow quaternary amineanion exchange column (20 mL bed volume equilibrated with MES buffer).The Akta protein purification system as described for the cationexchange step was used. Proteins were eluted with a 0-0.5 M NaCIgradient in MES buffer. The active enzymes were then exchanged into 50mM, pH 5.0 Sodium Acetate buffer before cation exchange chromatography.

7.1.16 Purification of Xvlanases

Xylanase purification was conducted in two stages:

Stage 1: Hydrophobic interaction chromatography (HIC). (NH₄)₂SO₄ wasadded to fermentation supernatant to achieve a final concentration of 1M. The separation column used was a Hiprep Phenyl (highsub), 16/10, 20mL. Buffer A: 20 mM sodium phosphate, pH 6.0. Buffer B: Buffer A +1 M(NH₄)₂SO₄. Stepwise Elution: 45% B; 0% B; water+10% glycerol.

Stage 2: Gel filtration (GF). Eluate of 0% B from the HIC column wascollected and loaded on a HiLoad 26/60 Superdex 75 prep grade (320 mL,GE Healthcare) column. The mobile phase was 20 mM sodium phosphate, pH6.8, containing 0.15 M NaCl. The elution profile is shown in FIG. 26.FIG. 27 shows the SDS-PAGE detection of the two step separation ofAfuXyn 5.

The Applied Biosystems BIOCAD® Vision and Amersham Pharmacia BiotechAKTA Explorer were used for protein purification of T. reesei XYN3.Approximately 150 mL of Xylanase 3, from an ultrafiltration concentrate,was loaded onto a Sephadex G-25M desalting column (total volume ˜525 mL)equilibrated in 10 mM TES buffer, pH 6.8. 300-400 mL of this desaltedsample was then loaded onto an anion-exchange column (high densityquaternary amine resin; Applied Biosystems Inc.). The bound protein wasthen eluted using a salt gradient between 0-1 M sodium chloride using8-column volumes of 25 mM TES buffer. Elution of the protein occurredbetween 0-250 mM sodium chloride, and was detected using 10% Bis-TrisNUPAGE® SDS-PAGE (Novex).

The eluted Xylanase 3-containing samples were concentrated to 10 mL withVivaspin 5,000 MWCO (molecular weight cut-off) membrane concentrators(Vivascience; GE Healthcare). The concentrate was then loaded onto aHigh Load 26/60 Superdex 200 (ID no 17-1071-01; GE Healthcare). Thecolumn was equilibrated with 25 mM TES buffer, pH 6.8, containing 100 mMsodium chloride, and the bound protein was eluted over 8-column volumesof the TES buffer with sodium chloride.

The purity of the eluted protein was assessed using 10% Bis-Tris NUPAGE®SDS-PAGE (Novex), and determined to be greater than 95% pure.

7.1.17 Purification of Fv43D

The ultrafiltration concentrate (UFC) of Fusarium verticillioides 43Dwas buffer exchanged and dialyzed against 50 mM Sodium Acetate buffer,pH 4.0, overnight. The dialyzed material was passed through a HiTrap 1mL column prepacked with sulfopropyl sepharose fast flow resin (GEHealthcare) designed for use with a syringe. The purified Fv43D waseluted with 50 mM Sodium Acetate buffer, pH 4.0, with 250 mM sodiumchloride. The UFC and Sodium Acetate buffer were pushed through thecolumn using a 5 mL syringe fitted with a GE Healthcare connector. Thepurified Fv43D was dialyzed overnight against 50 mM Sodium Acetatebuffer, pH 4.0. The purified protein was assayed by SDS-PAGE, HPLC, andmass spectroscopy to demonstrate homogeneity.

7.1.18 Purification of Fv51A

The ultrafiltration concentrate (UFC) of Fusarium verticillioides 51Awas buffer exchanged and dialyzed against 50 mM Sodium Acetate buffer,pH 5.0, overnight. The dialyzed material was passed through a RESOURCE15 6 mL column prepacked with methyl sulfonate media (GE Healthcare).The UFC was loaded at 1 mL/min against 50 mM Sodium Acetate buffer, pH5.0, and eluted at 5 mL/min against 50 mM Sodium Acetate buffer, pH 5.0,using a 0 to 250 mM sodium chloride gradient. The eluted fractions werecollected and assayed by SDS-PAGE. Fractions with purified Fv51A wereconcentrated using a 10,000 MWCO Vivaspin concentrator from SartoriusStemdim Biotech. The purified Fv51A was dialyzed against 50 mM SodiumAcetate buffer, pH 5.0, overnight. The purified protein was assayed bySDS-PAGE, H PLC, and mass spectroscopy to demonstrate homogeneity. TheAKTA Explorer 100 system from GE Healthcare was used for thepurification of Fv51A.

8. EXAMPLE 2: EXPRESSION OF INDIVIDUAL HEMICELLULASE GENES FROM VARIOUSSPECIES IN TRICHODERMA REESEI

8.1 Fusarium verticillioides qenes

The sequence for Fv51A was obtained by searching the Fusariumverticillioides genome in the Broad Institute database(www.broadinstitute.org/) for GH51 arabinofuranosidase homologs.

The following genes from Fusarium verticillioides were expressed inTrichoderma reesei: Fv3A, Fv43A, Fv43B, Fv43D, Fv51A, Fv3B, Fv43C,Fv39A, Fv43E, Fv30A, Fv30B, and Fv43F. Fv3A sequence was obtained bysearching for GH3 β-xylosidase homologs in Fusarium verticillioidesgenome. The annotated sequence lacked a signal sequence and the geneprediction program Augustus (augustus.gobics.de/) was used to identifyupstream sequence which contained a signal sequence. Sequences forFv39A, Fv43A, Fv43B, Fv43D, Fv43E, and Fv30A were obtained by searchingthe Fusarium verticillioides genome for GH39, GH30, and GH43β-xylosidase homologs.

Open reading frames of the hemicellulase genes of interest wereamplified by PCR using purified/extracted genomic DNA from Fusariumverticillioides as the template. The PCR thermocycler used was DNAEngine Tetrad 2 Peltier Thermal Cycler (BioRad Laboratories). The DNApolymerase used was PfuUltra II Fusion HS DNA Polymerase (Stratagene).The primers used to amplify the open reading frames were as follows:

Fv3A: Forward primer MH124 (SEQ ID NO: 52)(5′-CACCCATGCTGCTCAATCTTCAG-3′) Reverse primer MH125 (SEQ ID NO: 53)(5′-TTACGCAGACTTGGGGTCTTGAG-3′) Fv43A: Forward primer MH075(SEQ ID NO: 54) (5′-CACCATGTGGCTGACCTCCCCATT-3′) Reverse primer MH076(SEQ ID NO: 55) (5′-TTAGCTAAACTGCCACCAGTTGAAGTTG-3′)  Fv43B:Forward primer MH077 (SEQ ID NO: 56) (5′-CACCATGCGCTTCTCTTGGCTATTGT-3′)Reverse primer MH078 (SEQ ID NO: 57) (5′-CTACAATTCTGATTTCACAAAAACACC-3′)Fv43D: Forward primer MH081 (SEQ ID NO: 58)(5′-CACCATGCAGCTCAAGTTTCTG-3′) Reverse primer MH082 (SEQ ID NO: 59)(5′-CTAAATCTTAGGACGAGTAAGC-3′) Fv51A: Forward primer SK1159(SEQ ID NO: 60) (5′-CACCATGGTTCGCTTCAGTTCAATCCTAG-3′)Reverse primer SK1160 (SEQ ID NO: 61) (5′-CTAGCTAGAGTAAGGCTTTCC-3′)Fv39A: Forward: MH116 (SEQ ID NO: 62)(5′-CACCATGCACTACGCTACCCTCACCAC-3′) Reverse: MH117 (SEQ ID NO: 63)(5′-TCAAGTAGAGGGGCTGCTCACC-3′) Fv3B: Forward primer MH126(SEQ ID NO: 64) (5′-CAC CAT GAA ACT CTC TAG CTA CCT CTG-3′)Reverse primer MH127 (SEQ ID NO: 65)(5′-CTA CGA AAC TGT GAC AGT CAC GTT G-3′) Fv30A: Forward primer MH112(SEQ ID NO: 66) (5′-CAC CAT GCT CTT CTC GCT CGT TCT TCC TAC-3′)Reverse primer MH113 (SEQ ID NO: 67) (5′-TTA GTT GGT GCA GTG GCC ACG-3′)Fv30B: Forward primer MH114 (SEQ ID NO: 68)(5′-CAC CAT GAA TCC TTT ATC TCT CGG CCT TG-3′) Reverse primer MH115 (SEQ ID NO: 69) (5′-CAG CCC TCA TAG TCG TCT TCT TC-3′) Fv43C:Forward primer MH079 (SEQ ID NO: 70)(5′-CAC CAT GCG TCT TCT ATC GTT TCC-3′) Reverse primer MH080(SEQ ID NO: 71) (5′-CTA CAA AGG CCT AGG ATC AA-3′) Fv39A:Forward primer MH116 (SEQ ID NO: 72)(5′-CAC CAT GCA CTA CGC TAC CCT CAC CAC-3′) Reverse primer MH117(SEQ ID NO: 73) (5′-TCA AGT AGA GGG GCT GCT CAC C-3′) Fv43E:Forward primer MH147 (SEQ ID NO: 74)(5′-CAC CAT GAA GGT ATA CTG GCT CGT GG-3′) Reverse primer MH148(SEQ ID NO: 75) (5′-CTA TGC AGC TGT GAA AGA CTC AAC C-3′) Fv43F:Forward primer MH149 (SEQ ID NO: 76) (5′-CACCATGTGGAAACTCCTCGTCAGC-3′)Reverse primer MH150  (SEQ ID NO: 77)(5′-CTA ATA AGC AAC AGG CCA GCC ATT G-3′)

The forward primers included four additional nucleotides(sequences—CACC) at the 5′-end to facilitate directional cloning intopENTR/D-TOPO (Invitrogen, Carlsbad, Calif.) (FIG. 28). The PCRconditions for amplifying the open reading frames were as follows(except for Fv51A): Step 1: 94° C. for 2 min. Step 2: 94° C. for 30 sec.Step 3: 57° C. for 30 sec. Step 4: 72° C. for 30-45 sec. Steps 2, 3 and4 were repeated for an additional 29 cycles. Step 5: 72° C. for 2 min.For Fv51A, the following conditions were used: Step 1: 94° C. for 2 min.Step 2: 94° C. for 30 sec. Step 3: 56° C. for 30 sec. Step 4: 72° C. for45 sec. Steps 2, 3, 4 were repeated for an additional 25 cycles. Step 5:4° C. hold.

The PCR products of the corresponding hemicellulase open reading frameswere purified using a Qiaquick PCR Purification Kit (Qiagen, Valencia,Calif.). The purified PCR products were cloned into the pENTR/D-TOPOvector, transformed into TOP10 chemically competent E. coli cells(Invitrogen, Carlsbad, Calif.) and plated on LA plates with 50 ppmkanamycin. Plasmid DNA was obtained from the E. coli transformants usinga QIAspin plasmid preparation kit (Qiagen). Sequence data for the DNAinserted in the pENTR/D-TOPO vector was obtained using M13 forward andreverse primers (Sequetech, Mountain View, Calif.). A pENTR/D-TOPOvector with the correct DNA sequence of the corresponding hemicellulaseopen reading frame was recombined with the pTrex3gM destination vector(WO 05/001036, FIG. 29) using LR clonase reaction mixture (Invitrogen,Carlsbad, Calif.) according to the manufacturer's instructions. Theproduct of the LR clonase reaction was subsequently transformed intoTOP10 chemically competent E. coli cells which were then plated on LAcontaining 50ppm carbenicillin. The resulting expression vectors werepTrex3gM plasmids containing the corresponding hemicellulase openreading frames that resulted from the recombination event between theattR1 and attR2 sites of pTrex3gM and the attL1 and attL2 sites ofpENTR/D-TOPO; and the Aspergillus nidulans acetamidase selection marker(amdS). DNA of the expression vectors containing the correspondinghemicellulase open reading frames were isolated using a Qiagen miniprepkit and used for biolistic transformation of Trichoderma reesei spores.

Biolistic transformation of Trichoderma reesei with the pTrex3gMexpression vector containing the corresponding hemicellulase openreading frame was performed using the following protocol. Transformationof the Trichoderma reesei cellulase quad delete (Δcbh1, Δcbh2, Δeg1,Δeg2) strain by helium-bombardment was accomplished using a Biolistic®PDS-1000/He Particle Delivery System from Bio-Rad (Hercules, Calif.)following the manufacturer's instructions (see patent publications WO05/001036 and US 2006/0003408). Transformants were transferred to freshacetamide selection plates (see patent publication WO 2009114380).Stable transformants were inoculated into filter microtiter plates(Corning), containing 200 μL/well of Glycine Minimal media (6.0 g/Lglycine; 4.7 g/L (NH₄)₂SO₄; 5.0 g/L KH₂PO₄; 1.0 g/L MgSO₄.7H₂O; 33.0 g/LPIPPS; pH 5.5) with post sterile addition of ˜2% glucose/sophorosemixture (U.S. Pat. No. 7,713,725) as the carbon source, 10 mL/L of 100g/L of CaCl₂, 2.5 mL/L of T. reesei trace elements (400×): 175 g/LCitric acid anhydrous; 200 g/L FeSO₄.7H₂O; 16 g/L ZnSO₄.7H₂O; 3.2 g/LCuSO₄.5H₂O; 1.4 g/L MnSO₄.H₂O; 0.8 g/L H₃Bo₃). Transformants were grownin liquid culture for 5 days in an O₂-rich chamber housed in a 28° C.incubator. The supernatant samples from the filter microtiter plate werecollected on a vacuum manifold. Supernatant samples were run on 4-12%NuPAGE gels (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions to check for expression. The gel was stainedwith Simply Blue stain (Invitrogen, Carlsbad, Calif.). Purification ofGH43, GH51 and GH3 enzymes from T. reesei fermentations was performed bycation exchange chromatography as described in Example 1.

8.2 Genes from Other Species

Pf43A and Pf51A sequences were obtained by sequencing of the Penicilliumfuniculosum genome. The queries used for the searches of the P.funiculosum genome were other fungal GH43 and GH51 homologs available inthe public genomes. A gene prediction program named Augustus(augustus.gobics.de/) was used to verify the intron sequences, alongwith the start codon. Fv43D sequence was used to query the Gibberellazeae (Fusarium graminearum) and Fusarium oxysporum genomes available inthe Broad Institute database and retrieve sequences for Gz43A and Fo43Arespectively. The genes for Gz43A and Fo43A were synthesized by GeneArt(Geneart GmbH, Regensburg, Germany) with the CBH1 signal sequence inplace of the native signal sequence. Neither gene contained introns. ThePf51A gene was codon-optimized and synthesized by GeneArt with the CBH1signal sequence in place of the native signal sequence.

Pf43A and Pf51A were cloned and expressed in Trichoderma reesei usingthe following primers:

Pf43A: MH151 (SEQ ID NO: 78) (5′-CACCATGCTTCAGCGATTTGCTTATATTTTACC-3′)Pf43A: MH152 (SEQ ID NO: 79) (5′-TTATGCGAACTGCCAATAATCAAAGTTG-3′)Pf51A: SK1168 (SEQ ID NO: 80) (5′-CACCATGTACCGGAAGCTCGCCGTG-3′)Pf51A: SK1169: (SEQ ID NO: 81) (5′-CTACTCCGTCTTCAGCACAGCCAC-3′)

Three genes were cloned from Aspergillus fumigatus for expression inTrichoderma reesei: GH11 xylanase 2 (AfuXyn2), GH11 xylanase 5(AfuXyn5), and GH43 Af43A. The primers for AfuXyn2, AfuXyn5, and Af43Acloning primers are shown below:

AfuXyn2: A.fumi-Q4WG11-F: (SEQ ID NO: 82)(5′-CCGCGGCCGCACCATGGTTTCTTTCTCCTACCTGCTGCTG-3′) A.fumi-Q4WG11-R:(SEQ ID NO: 83) (5′-CCGGCGCGCCCTTACTAGTAGACAGTGATGGAAGCAGATCCG-3′)AfuXyn5: A.fumi-Q4WFZ8-F: (SEQ ID NO: 84)(5′-CCGCGGCCGCACCATGATCTCCATTTCCTCGCTCAGCT-3′) A.fumi-Q4WFZ8-R:(SEQ ID NO: 85) (5′-CCGGCGCGCCCTTATCACTTGGATATAACCCTGCAAGAAGGTA-3′)Af43A: SK1203: (SEQ ID NO: 86) (5′-CACCATGGCAGCTCCAAGTTTATCC-3′) SK1204(SEQ ID NO: 87) (5′-TCAGTAGCTCGGGACCACTC-3′)

The methods used for cloning and expression of all these genes weresimilar to the procedure described for cloning of the Fusarium genes.Additional genes including those listed in FIG. 68, were cloned in asimilar manner.

9. EXAMPLE 3: TESTING FOR ACTIVITY OF NOVEL HEMICELLULASES ON SYNTHETICSUBSTRATES

The activities of Fv3A, Fv43A, Fv43B, Fv43D and a number of otherproteins in, for example, FIG. 69, were tested on synthetic substratespNPX and pNPA as described in Synthetic substrate activity assay inExample 1. T. reesei Bxl1 was used in at 0.7 g/L. The other enzymesamples, and the Quad-delete host control, were added by volume fromgrowth cultures (microtiter plate or shake flask scale). Therefore, theabsolute activity cannot be compared across samples but is an indicationof an active expressed protein with the relative pNPX and pNPA activityshown in FIG. 69. Activity on para-nitrophenyl substrates is not used asa predictor of performance in biomass saccharification.

10. EXAMPLE 4: HYDROLYSIS OF PRETREATED CORNCOB BY CELLULASE ANDHEMICELLULASE PREPARATIONS

10.1 Saccharification performance of expressed proteins

The saccharification performance of expressed proteins as additions toan enzyme mixture with an L-α-arabinofuranosidase deficiency wasevaluated. L-α-arabinofuranosidase candidates were evaluated in a 4-daycob saccharification assay by addition to an enzyme mixture ofAccellerase® 1500/T. reesei Xyn3/Fv3A. The screen was conducted asdescribed in the corncob Saccharification Assay (Example 1) with thefollowing enzymes and amounts/concentrations:

Accellerase® 1500, TP (Total Nitrogen) 54.2 mg/mL

Trichoderma reesei Xyn3, 2.9 mg/mL TP (purified)

Fv3A, 3.2 mg/mL TP (purified)

Fv51A, 7.8 mg/mL TP (purified)

Mg51A, 6.8 mg/mL TP (TCA/BCA)

At51A, 6.7 mg/mL TP (TCA/BCA)

Pt51A, 3.3 mg/mL TP (TCA/BCA)

Ss51A, 3.0 mg/mL TP (TCA/BCA)

Vd51A, 6.8 mg/mL TP (TCA/BCA)

Cg51B, 3.6 mg/mL TP (TCA/BCA)

Af43A, 2.6 mg/mL TP (TCA/BCA)

Pf43A, 2 mg/mL TP (TCA/BCA)

Fv43E, 1.37 mg/mL TP (TCA/BCA)

The total protein (TP) of purified samples was determined by A280 unlessotherwise noted. The total protein of unpurified samples was determinedby BCA according to manufacturer instructions, unless otherwise noted.Accellerase® 1500 was added at 20 mg protein/g cellulose; Trichodermareesei Xyn3 was added at 5 mg protein/g cellulose; Fv3A was added at 5mg protein/g cellulose. Fv51A, Mg51A, At51A, Pt51A, Ss51A, Vd51A, Cg51B,Af43A, Pf43A, or Fv43E were added at 1, 3, and 5 mg protein/g cellulose.Following 4 days incubation, 50° C., 200 rpm, the assay plate wasquenched and analyzed by HPLC for soluble sugars.

Enzymes were found that enhanced glucose or xylose or arabinose yield,or reduced cellobiose or xylobiose concentration in this enzyme mixture.Results are shown in FIGS. 30A and 30B.

The saccharification performance of expressed proteins was alsoevaluated as additions to the enzyme mixture with anL-α-arabinofuranosidase and β-xylosidase deficiency. β-xylosidasecandidates were evaluated in a 3 day cob saccharification assay byaddition to an enzyme mixture of Accellerase® 1500/T. reesei Xyn3. Thescreen was conducted as described in the corncoob saccharification assay(Example 1) with the following enzymes and amounts/concentrations:

Accellerase® 1500, TP (total nitrogen) 54.2 mg/mL

Trichoderma reesei Xyn3, 2.9 mg/mL (purified)

Fv3A, 3.2 mg/mL (purified)

Fv43D, 6.8 mg/mL (purified)

Pf43A, 2 mg/mL TP (BCA)

Pf43B, 2.7 mg/mL TP (BCA)

Fv43E, 1.37 mg/mL TP (BCA)

Fv43F, 2.8 mg/mL TP (BCA)

Fv30A, 2.7 mg/mL TP (BCA)

Accellerase® 1500 was added at 17.9 mg protein/g cellulose; T. reeseiXyn3 was added at 5 mg protein/g cellulose. Fv3A, Fv43D, Pf43A, Pf43B,Fv43E, Fv43F, or Fv30A were added at 1, 3, and 5 mg protein/g cellulose(Fv43E was only added at 1 and 3 mg/g). Following 3 days incubation, 50°C., 200 rpm, the assay plate was quenched and analyzed by HPLC forsoluble sugars. Results are shown in FIG. 31. Enzymes were found thatenhanced glucose, or xylose or arabinose yield, or reduced cellobiose orxylobiose concentration in this enzyme mixture.

The saccharification performance of expressed proteins was alsoevaluated as additions to the enzyme mix with a xylanase deficiency.During the construction of the Trichoderma reesei integrated expressionstrains (described in Example 9 below), one T. reesei strain (strain #44) was isolated that over-expressed Bgl1, Fv3A, Fv51A, Fv43D proteinsbut did not over-express endo-xylanase. This strain was used as thebackground to which candidate xylanases were added for performancescreening. Endo-xylanase candidates were evaluated in a 3 day cobsaccharification assay by addition to the enzyme products from strain#44. The screen was conducted as described in the corncobsaccharification assay (Example 1) with the following enzymes andamounts/concentrations:

Strain #44 enzyme product 78.6 mg/mL TP (modified Biuret)

Trichoderma reesei Xyn3 2.9 mg/mL TP (purified)

AfuXyn2 3.3 mg/mL TP (purified)

AfuXyn3 5.8 mg/mL TP (purified)

AfuXyn5 14.8 mg/mL TP (purified)

PfuXyn1 1.9 mg/mL TP (purified)

SspXyn1 1.2 mg/mL TP (purified)

The enzyme composition produced by Strain #44 was added at 20 mgprotein/g cellulose; candidate xylanase enzymes were added at 3 and 7 mgprotein/g cellulose. Following 3 days incubation, at 50° C., 200 rpm,the assay plate was quenched and analyzed by HPLC for soluble sugars.Enzymes were found that enhanced xylose, or glucose, or arabinose yield,or reduced cellobiose or xylobiose concentration in this enzyme mixture.Results are shown in FIG. 32.

The saccharification performance of expressed Fv51A and Pa51A proteinsin enzyme mixtures with L-α-arabinofuranosidase deficiency was evaluatedand compared. Fv51A and Pa51A were evaluated in a 3 day cobsaccharification assay by addition to an enzyme mixture of Accellerase®1000/T. reesei Xyn2/Bxl1 or Fv3A. The screen was conducted as describedin the corncob saccharification assay (Example 1) with the followingenzymes and amounts/concentrations:

Accellerase® 1000, 60.6 mg/mL TP (total nitrogen)

Trichoderma reesei Xyn2 4.1 mg/mL TP (purified)

Trichoderma reesei Bxl1 69 mg/mL TP (TCA/total nitrogen)

Fv3A 65 mg/mL TP (TCA/total nitrogen)

Fv51A 43 mg/mL TP (TCA/BCA)

Pa51A 85.4 mg/mL TP (TCA/total nitrogen)

Accellerase® 1000 was added at 20 mg protein/g cellulose; Trichodermareesei Xyn2 was added at 5 mg protein/g cellulose; Trichoderma reeseiBxl1 or Fv3A was added at 5 mg protein/g cellulose. Fv51A was added at 5mg protein/g cellulose. Pa51A was added at 1, 2, or 5 mg protein/gcellulose. Following 3 days incubation, at 50° C., 200 rpm, the assayplate was quenched and analyzed by HPLC for soluble sugars. Enzymecombinations were found that enhanced xylose, or glucose, or arabinoseyield, or reduced cellobiose or xylobiose concentration in this enzymemixture. Results are shown in FIG. 33.

Fv51A and Pa51A also were evaluated in a 3 day cob saccharificationassay by addition to an enzyme mixture of Accellerase® 1000/ Trichodermareesei Xyn2. Purified Fv51A (29 mg/mL TP) and Pa51A (29 mg/mL) were usedin this part of the study. Accellerase® 1000 was added at 17.5 mgprotein/g cellulose; Trichoderma reesei Xyn2 was added at 4.4 mgprotein/g cellulose. Fv51A was added at 4.4 mg protein/g cellulose.Pa51A was added at 0.9, 1.8, and 4.4 mg protein/g cellulose. Following 3days incubation, at 50° C., 200 rpm, the assay plate was quenched andanalyzed by HPLC for soluble sugars. Enzyme combinations were found thatenhanced xylose, or glucose, or arabinose yield in this enzyme mixture.Results are shown in FIG. 34.

The saccharification performance of expressed proteins was alsoevaluated as additions to the enzyme mix with β-xylosidase deficiency.β-xylosidase candidates were evaluated in a 3 day cob saccharificationassay by addition to an enzyme mixture of Accellerase® 1000/ Trichodermareesei Xyn2/Fv51A. The screen was conducted as described in the corncobsaccharification assay (Example 1) with the following enzymes andamounts/concentrations:

Accellerase® 1000, TP (TCA/Total nitrogen) 60.6 mg/mL

Trichoderma reesei Xyn2, 4.1 mg/mL TP (purified)

Fv3A, 65 mg/mL TP (TCA/Total nitrogen)

Fv3B, 62.9 mg/mLTP (TCA/Total nitrogen)

Fv39A, 47.5 mg/mL TP (TCA/Total nitrogen)

Fv30B, 62.9 mg/mL TP (TCA/Total nitrogen)

Fv51A, 43 mg/mL TP (TCA/BCA)

Accellerase® 1000 was added at 20 mg protein/g cellulose; Trichodermareesei Xyn2 was added at 5 mg protein/g cellulose; Fv51A was added at 5mg protein/g cellulose. Fv39A, Fv30B, Fv3A, or Fv3B were added at 1, 2,or 5 mg protein/g cellulose. Following 3 days incubation, at 50° C., 200rpm, the assay plate was quenched and analyzed by HPLC for solublesugars. Enzymes and combinations were found that enhanced glucose orxylose or arabinose yield, or reduced cellobiose or xylobioseconcentration in this enzyme mixture, with or without anotherβ-xylosidase (T. reesei Bxl1). Results are shown in FIGS. 35A-35C.

10.2 Activity of Candidate Endo-xylanases with Birchwood Xylan

The activity of candidate endo-xylanases was evaluated using birchwoodxylan as a substrate using the following assay. Ninety microliters of a1% (wt/vol) birchwood xylan (Sigma X0502) stock solution was added towells in a 96-well microtiter plate and pre-incubated at 50° C. for 10min. Enzyme dilutions and xylose standards were added (10 μL) to themicrotiter plate and the plates incubated at 50° C. for 10 min.Meanwhile, 100 μL of DNS solution was added to PCR tubes. Following the10-min incubation, 60 pL of the enzyme reaction was transferred to thePCR tubes containing the DNS solution. The tubes were incubated in athermocycler at 95° C. for 5 min, and then cooled to 4° C. One hundredmicroliters of the reaction mixture was transferred to a 96-well plate,and absorbance at 540 nm was measured. A xylose standard curve wasgenerated and used to calculate the activity. One xylanase unit isdefined as the amount of enzyme required to generate 1 μmole of xylosereducing sugar equivalents per minute under the conditions of the assay.Results are shown in FIG. 70.

10.3 Enzyme Hydrolysis of Arabinoxylan Oligomers from SaccharifiedCorncob

In this study, enzyme hydrolysis of the arabinoxylan oligomers remainingafter digestion of dilute ammonia pretreated corncob with cellulase andhemicellulase preparations was monitored. Preparation of crude oligomersis described in Example 1. Total oligomer sugars were determined by HPLC(see Example 1) after acid hydrolysis of the crude oligomers with 2%(v/v) sulfuric acid in a sealed vial at 121° C. for 30 min. The sugarconcentrations were corrected for a small amount of sugar degradation asdetermined by control samples of known sugar mixtures treated by thesame procedure. The concentration of total sugars in the crude oligomerpreparation determined by this method was 45 g/L glucose, 168 g/Lxylose, and 46 g/L arabinose. When accounting for monomer sugar presentin crude oligomers before acid hydrolysis, 86% of the glucose, 90% ofthe xylose, and 43% of the arabinose was present in oligomeric form.Various β-xylosidases, arabinofuranosidases, and mixtures thereof weretested for increased conversion of arabinose monomer from the crudeoligomers preparation. The crude oligomer preparation was diluted20-fold to 12 g/L oligomers in 50 mM Sodium Acetate buffer, pH 5.0, andmaintained at 50° C. in a heating block in capped 1.5 mL Eppendorftubes. Enzymes were added at final concentrations of 0.06-0.09 g/L andincubated for 24 h to reach completion. Samples were then removed forHPLC analysis of momomer sugars as described in Example 1. The resultsare listed in FIG. 71 as % conversion to monomer sugar based on totalsugar as determined by acid hydrolysis.

To obtain the highest yields (44-71%) of arabinose from the remainingarabinoxylan oligomers in the crude oligomer mix, the data in FIG. 71show that binary combinations of Fv3A+Fv51A, Fv3A+Fv43B, and Fv43A+Fv43Bprovide the best results. From the sequence families and activity onartificial substrates, it is deduced that Fv3A is a β-xylosidase andFv51A is an L-α-arabinofuranosidase.

It is known that arabinose sugars in arabinoxylan from corncob arefrequently linked to xylose at both the 2 and 3 carbon positions of thearabinose sugar. Thus the activity of Fv3A is likely to hydrolyze thexyl(1-2)ara linkage which then makes available the ara(1-3) xyl linkageto hydrolysis by the L-α-arabinofuranosidase. Of the β-xylosidasestested only Fv3A and Fv43A appeared to have this activity. Also in thisscreening only Fv43B appeared to have L-α-arabinofuranosidase activityamong the Family 43 members from Fusarium verticillioides. Results areshown in FIG. 71.

11. EXAMPLE 5: SUBSTRATE RANGE OF B-XYLOSIDASES FOR EFFECTIVE CORNCOBHYDROLYSIS

In this example, the substrate range of 3 β-xylosidases and theirrelation to effective conversion of corncob xylooligomers to monomersugars were determined. Preparation of corncob hydrolysate containingoligomeric sugars and assay of monomer sugars was performed as describedin Example 1. The proton NMR spectra of oligomeric sugars with degree ofpolymerization (DP) greater than 2 as separated by size exclusionchromatography on Bio-Gel P2 were determined (FIG. 36 and FIG. 37). Thespectra of oligomers before enzyme treatment is labeled “MD07 oligomers”in the bottom panel of FIG. 36 and spectra of the same oligo containingfractions after enzyme treatments are in the remaining panels of FIG. 36and FIG. 37 labeled with the treatment enzyme.

The Bio-gel P2 fractions containing oligomers of greater than DP2 (5-10mg) were lyophilized then dissolved in 0.7 mL of a D₂O solutioncontaining 0.5 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) asinternal standard. Aliquots of 0.55 mL were used for the NMR samples inorder to optimize suppression of the residual water peak. Standardversions of the Varian 2D correlation pulse sequences were used, withoptimization of the ¹³C spectral width for the heteronuclearexperiments. Spectra were acquired on a Varian Unity Inova using a highsensitivity cryoprobe operating at 500 MHz. Structure elucidation wasdone by identifying the correlations that characterize the spin-systemfor the individual sugar residues and then identifying theinter-glycosidic correlations.

The arabinose containing oligomers as determined by NMR, are dominatedby one or more branched structures in which arabinose is linked β-1→3 toa xylose residue in a polymer fragment. The arabinose residue in theresulting branch is further substituted by a xylose residue linked β-1→2to the arabinose. Little arabinose without this second substitution ispresent in the remaining xylo-oligomers. T. reesei Bxl1 is not veryeffective at cleaving the furthest out xylose 1→2 to arabinose bond asevidenced by the remaining signals at 5.5 to 5.55 ppm in the spectra.The combination of the Fv43A that is effective on longer chain xyloseoligomers and the Fv43B L-α-arabinofuranosidase removes most but not allof the branched species with signals in the 5.5 ppm range and unliketreatment with the T. reesei Bxl1, leaves none of the signal at 5.35that is attributable to remaining arabinose branched to xylose oligomer.

The anomeric proton region of the spectra of the remainingxylo-oligomers after treatment with the Fv51A alone or in combinationwith the Fv3A show different results. The L-α-arabinofuranosidase aloneis not effective at reducing the complexity of signals in the region.The Fv3A β-xylosidase removes essentially all of the xylose subtendingthe arabinose branch leaving mostly simply linked arabinose 1→3 xyloseoligomer structures. Addition of the L-α-arabinofuranosidase to theβ-xylosidase results in a fairly complete conversion to monomer sugarsas evidenced by the increase in signal coming from the α and β anomericprotons of reducing sugars.

It can thus be concluded that the increased effectiveness of oneβ-xylosidase over another is likely to be due to the substrate range interms of the structural complexity of the aglycon allowable assubstrate.

12. EXAMPLE 6: SEQUENCE COMPARISON AND CRITICAL RESIDUES IN DETERMININGSUBSTRATE RANGE IN GH3 B-XYLOSIDASES

The T. reesei β-xylosidase (Bxl1) is 61% similar to the F.verticillioides ortholog (Fv3A) and shares 42% sequence identity asshown in the alignment of FIG. 38. Fv3A has much broader substrate rangethan Bxl1, which is likely to be attributable to the non-conserved aminoacids among the two proteins.

13. EXAMPLE 7: DETERMINATION OF DEFINED HEMICELLULASE ACTIVITY FORCORNCOB HYDROLYSIS TO MONOMER SUGARS

Pretreated corncob as described in Example 1 was used as water slurryadjusted to about pH 5 with H₂SO₄ in water at 18.6 g dry corncobsolids/100 g of total slurry. 0.78 g of the slurry was added to 4 mLglass vials and sufficient pH 5.0, 50 mM Sodium Acetate buffer was addedto give a total reaction weight of 1.06 g after the desired enzymeadditions. The hemicellulases were added as the purified preparationsdescribed in Example 1. Supernatant from the quad deleted T. reeseistrain (Quad in FIG. 72A, FIG. 72B, and FIG. 73) is the concentrate ofbackground proteins expressed by the T. reesei strain deleted in 4 majorcellulase activities (described in WO 05/001036). Accellerase® 1000 is awhole cellulase mixture with high β-glucosidase activity. The vials wereincubated at 230 rpm in an orbital shaker at 48° C. for 72 h then 2 mLof water was added. A sub-sample was taken and further diluted,centrifuged and filtered for HPLC analysis for monomer sugars asdescribed in Example 1. Experimental results defining useful amounts ofdefined hemicellulase activity for hydrolyzing pretreated corncob tomonomer sugars is shown in FIG. 72A, and FIG. 72B.

The results from the design-of-experiments (DoE)were fit to a surfacemodel and used to determine best ratios of the 7 enzyme components forbest yield of glucose, xylose and arabinose at the two total proteinconcentrations tested. Results of the ratios for the seven enzymecomponents are shown in FIG. 73.

Another exploration of the ratios (FIG. 74) was conducted includingFv3A, and again including Fv43D and holding that activity constant at alow level. The reaction set up and reaction conditions were identical tothose described for the full DoE experiment.

Reactions (run numbers) 20, 21, and 22 contain only the whole cellulaseenzyme mix and at the 21 mg/g of glucan loading monomerized about 48% ofthe glucose present in the cob and 24% of the xylose. Addition of theendoxylanase, Trichoderma reesei Xyn3 allowed decrease of the wholecellulase protein load while retaining about the same glucose monomeryield and increased the xylose monomer yield to about 40% (run# 1). Allthe combinations that gave arabinose yields of above 40% required thecombination of Fv43D, Fv43A and Fv43B or Fv51A or of Fv3A and Fv51A orFv43B. Those combinations also tended to have highest release of xyloseto monomer sugar.

Another set of reactions aimed at refining the required mix ofhemicellulases was run holding both the loading of whole cellulaseconstant and the loading of the endoxylanase constant. Accellerase® 1000whole cellulase preparation was held constant at 12 mg/g glucan andpurified T. reesei Xyn3 endoxylanase was held at 6 mg/g xylan. Fv51A wasthe only L-α-arabinofuranosidase in the mixture, at different doses.Other reaction conditions remained the same but the hydrolysate wasanalyzed by size exclusion chromatography as described in Example 1. Thequantitation of individual sugars was performed by peak area only andthe results are shown in FIG. 75.

All combinations released similar amounts of glucose and about the sameamount of total soluble xylose. The degree of reduction to monomerxylose varied by treatment. Without added activity to convert oligomerto monomer about 50% of the solubilized xylose remained oligomericunless at least a β-xylosidase was added. Fv3A at 2 mg Fv3A protein/gxylan reduced >DP2 oligomers to 3.6 mg/mL. Addition of 2 mg Fv51Aprotein/g xylan to the 2 mg Fv3A protein/g xylan further decreasedthe >DP2 oligomers to 1.5 mg/mL. About 2 mg of Fv51A/g xylan appeared tobe sufficient to reduce the >DP2 oligomers to a minimum when therequired 2 mg/g Fv3A was present (FIG. 75).

A mix of 6 mg/g xylan T. reesei Xyn3, 2 mg/g Fv3A and either 1 or 2 mg/gFv51A is a suitable loading to reduce total cob arabinoxylan to monomersugars. The addition of Fv43D to the mix aids in taking the xylobiose orother DP2 oligomers to monomer. Arabinose hydrolysis to monomer was notmeasured in this experiment.

14. EXAMPLE 8: EFFECTIVENESS OF HEMICELLULASES AT PRODUCING MONOMERSUGARS FROM CORNCOB

In this example, the effectiveness of a set of purified hemicellulaseactivities at producing monomer xylose and arabinose sugars when actingalone on diluted ammonia pretreated corncob is demonstrated. Threemixtures (Mixes A, B, & C) of purified hemicellulases were prepared andused to hydrolyze hemicellulose in pretreated cob in 1 g total, 14%solids reactions prepared as in Example 1 and run under the conditionsdescribed in Example 1. Monomer sugars were analyzed by HPLC asdescribed in Example 1 after 72 h of reaction and the amounts obtainedare shown in FIG. 76.

-   -   Mix A: 6 mg Trichoderma reesei Xyn3; 4 mg Fv3A; 1 mg Fv51A/g        xylan    -   Mix B: 6 mg Trichoderma reesei Xyn3; 1 mg Fv43D; 3 mg Fv43A; 3        mg Fv43B/g xylan    -   Mix C: 6 mg Trichoderma reesei Xyn3; 3 mg Fv3A; 1 mg Fv43D; 1 mg        Fv51A/g xylan

In this experiment the defined hemicellulase sets yielded slightly lessmonomer sugar than seen in earlier experiments which included activitiesto solubilize cellulose. The yields were still greater than those seenwith endoxylanase-only addition to whole cellulase preparations. Thehemicellulase activities are effective in taking xylan to monomer.

The same set of mixtures was used on hemicellulose preparations madefrom corncob, total stover from grain sorghum, switchgrass and sugarcane bagasse using the procedures in the general methods in accordancewith Example 1. Stock suspensions of each hemicellulose preparation at100 mg/mL in 50 mM pH 5.0 Sodium Acetate buffer were made and the pH waschecked. Each of them was diluted to 10 mg preparation per mL with more50 mM acetate buffer. Aliquots of each enzyme mixture were added to 100μL of the 10 mg/mL suspension and the reactions were run in duplicate,incubated at 48° C. for 6 h with agitation. Reactions were diluted with100 μL of water, centrifuged and filtered before HPLC analysis formonomer sugars as described in Example 1. 200 μL of each hemicellulosesuspension was diluted with 200 μL of 0.8 N H₂SO₄, autoclaved at 121° C.for 30 min on liquid cycle then filtered and sugars analyzed by HPLC asdescribed in Example 1. Results shown in FIG. 77 are reported as theaverage monomer sugar released by the enzyme mixture as a percentage ofthe acid hydrolysable sugar present in the reaction.

Mixes A and C performed well on hemicellulose from cob and as seen inother experiments on whole pretreated cob. Mixtures containing Fv3Aincrease conversion of arabinose to monomer and give a slight advantagein conversion of xylose to monomer. All 3 mixtures work well onhemicellulose purified from other monocots. The mixing of oneendoxylanase, either one or two β-xylosidases, one of which hassubstrate specificity beyond two or three xylose units linked β 1→4 andan L α-arabinofuranosidase results in an effective hemicellulase blendagainst monocot hemicellulose.

15. EXAMPLE 9: CONSTRUCTION OF THE INTEGRATED EXPRESSION STRAIN OFTRICHODERMA REESEI

An integrated expression strain of Trichoderma reesei was constructedthat co-expressed five genes: T. reesei β-glucosidase gene bgl1, T.reesei endoxylanase gene xyn3, F. verticillioides β-xylosidase genefv3A, F. verticillioides β-xylosidase gene fv43D, and F. verticillioidesα-arabinofuranosidase gene fv51A.

The construction of the expression cassettes for these different genesand the transformation of T. reesei strain are described below.

15.1 Construction of the β-glucosidase Expression Cassette

The N-terminal portion of the native T. reesei β-glucosidase gene bgl1was codon optimized by DNA 2.0 (Menlo Park, USA). This synthesizedportion comprised of the first 447 bases of the coding region. Thisfragment was PCR amplified using primers SK943 and SK941. The remainingregion of the native bgl1 gene was PCR amplified from a genomic DNAsample extracted from T. reesei strain RL-P37, using primer SK940 andSK942. These two PCR fragments of the bgl1 gene were fused together in afusion PCR reaction, using primers SK943 and SK942:

Forward Primer SK943: (SEQ ID NO: 88)(5′-CACCATGAGATATAGAACAGCTGCCGCT-3′) Reverse Primer SK941:(SEQ ID NO: 89)) (5′-CGACCGCCCTGCGGAGTCTTGCCCAGTGGTCCCGCGACAG-3′)Forward Primer SK940: (SEQ ID NO: 90)(5′-CTGTCGCGGGACCACTGGGCAAGACTCCGCAGGGCGGTCG-3′) Reverse Primer SK942:(SEQ ID NO: 91) (5′-CCTACGCTACCGACAGAGTG-3′)

The resulting fusion PCR fragments were cloned into the Gateway ® Entryvector pENTR™/D-TOPO®, and transformed into E. coli One Shot® TOP10Chemically Competent cells (Invitrogen) resulting in the intermediatevector, pENTRY-943/942 (FIG. 39). The nucleotide sequence of theinserted DNA was determined. The pENTRY-943/942 vector with the correctbgl1 sequence was recombined with pTrex3g using a LR clonase® reactionprotocol outlined by Invitrogen. The LR clonase reaction mixture wastransformed into E. coli One Shot® TOP10 Chemically Competent cells(Invitrogen), resulting in the final expression vector, pTrex3g 943/942(FIG. 40). The vector also contains the Aspergillus nidulans amdS geneencoding acetamidase as a selectable marker for transformation of T.reesei. The expression cassette was PCR amplified with primers SK745 andSK771 to generate product for transformation of the strain, using theelectroporation method described in WO 08153712.

Forward Primer SK771: (SEQ ID NO: 94) (5′-GTCTAGACTGGAAACGCAAC-3′)Reverse Primer SK745: (SEQ ID NO: 95) (5′-GAGTTGTGAAGTCGGTAATCC-3′)

15.2 Construction of the Endoxylanase Expression Cassette

The native T. reesei endoxylanase gene xyn3 was PCR amplified from agenomic DNA sample extracted from T. reesei, using primers xyn3F-2 andxyn3R-2.

Forward Primer xyn3F-2: (SEQ ID NO: 94)(5′-CACCATGAAAGCAAACGTCATCTTGTGCCTCCTGG-3′) Reverse Primer (xyn3R-2):(SEQ ID NO: 95) (5′-CTATTGTAAGATGCCAACAATGCTGTTATATGCCGGCTTGGGG-3′)

The resulting PCR fragments were cloned into the Gateway® Entry vectorpENTR™/D-TOPO®, and transformed into E. coli One Shot® TOP10 ChemicallyCompetent cells (Invitrogen) resulting in the intermediate vector,pENTR/Xyn3 (FIG. 41). The nucleotide sequence of the inserted DNA wasdetermined. The pENTR/Xyn3 vector with the correct xyn3 sequence wasrecombined with pTrex3g using a LR clonase® reaction protocol outlinedby Invitrogen. The LR clonase reaction mixture was transformed into E.coli One Shot® TOP10 Chemically Competent cells (Invitrogen), resultingin the final expression vector, pTrex3g/Xyn3 (FIG. 42). The vector alsocontains the Aspergillus nidulans amdS gene encoding acetamidase as aselectable marker for transformation of T. reesei. The expressioncassette was PCR amplified with primers SK745 and SK822 to generateproduct for transformation of the strain, using the electroporationmethod.

Forward Primer SK745: (SEQ ID NO: 96) (5′-GAGTTGTGAAGTCGGTAATCC-3′)Reverse Primer SK822: (SEQ ID NO: 97) (5′-CACGAAGAGCGGCGATTC-3′)

15.3 Construction of the β-xylosidase Fv3A Expression Cassette

The F. verticilloides β-xylosidase fv3A gene was amplified from a F.verticilloides genomic DNA sample using the primers MH124 and MH125.

Forward Primer MH124: (SEQ ID NO: 98)(5′-CAC CCA TGC TGC TCA ATC TTC AG-3′) Reverse Primer MH125:(SEQ ID NO: 99) (5′-TTA CGC AGA CTT GGG GTC TTG AG-3′)

The PCR fragments were cloned into the Gateway ® Entry vectorpENTR™/D-TOPO®, and transformed into E. coli One Shot® TOP10 ChemicallyCompetent cells (Invitrogen) resulting in the intermediate vector,pENTR-Fv3A (FIG. 43). The nucleotide sequence of the inserted DNA wasdetermined. The pENTRY-Fv3A vector with the correct fv3A sequence wasrecombined with pTrex6g using a LR clonase® reaction protocol outlinedby Invitrogen. The LR clonase reaction mixture was transformed into E.coli One Shot® TOP10 Chemically Competent cells (Invitrogen), resultingin the final expression vector, pTrex6g/Fv3A (FIG. 44). The vector alsocontains a chlorimuron ethyl resistant mutant of the native T.reeseiacetolactate synthase (a/s) gene, designated a/sR, which is usedtogether with its native promoter and terminator as a selectable markerfor transformation of T. reesei (WO2008/039370 A1). The expressioncassette was PCR amplified with primers SK1334, SK1335 and SK1299 togenerate product for transformation of T. reesei, using theelectroporation method (see, e.g., WO2008153712 A2).

Forward Primer SK1334: (SEQ ID NO: 100) (5′-GCTTGAGTGTATCGTGTAAG-3′)Forward Primer SK1335: (SEQ ID NO: 101) (5′-GCAACGGCAAAGCCCCACTTC-3′)Reverse Primer SK1299: (SEQ ID NO: 102)(5′-GTAGCGGCCGCCTCATCTCATCTCATCCATCC-3′)

15.4 Construction of the β-xylosidase Fv43D Expression Cassette

For the construction of the F. verticilloides β-xylosidase Fv43Dexpression cassette, the fv43D gene product was amplified fromF.verticilloides genomic DNA using the primers SK1322 and SK1297. Aregion of the promoter of the endoglucanase gene egl1 was PCR amplifiedfrom T. reesei genomic DNA extracted from strain RL-P37, using theprimers SK1236 and SK1321. These two PCR amplified DNA fragments weresubsequently fused together in a fusion PCR reaction using the primersSK1236 and SK1297. The resulting fusion PCR fragment was cloned intopCR-Blunt II-TOPO vector (Invitrogen) to give the plasmid TOPOBlunt/Pegll-Fv43D (FIG. 45) and E. coli One Shot® TOP10 ChemicallyCompetent cells (Invitrogen) were transformed using this plasmid.Plasmid DNA was extracted from several E.coli clones and confirmed byrestriction digest.

Forward Primer SK1322: (SEQ ID NO: 103)(5′-CACCATGCAGCTCAAGTTTCCTGTC-3′) Reverse Primer SK1297:(SEQ ID NO: 104) (5′-GGTTACTAGTCAACTGCCCGTTCTGTAGCGAG-3′)Forward Primer SK1236: (SEQ ID NO: 105)(5′-CATGCGATCGCGACGTTTTGGTCAGGTCG-3′) Reverse Primer SK1321:(SEQ ID NO: 106) (5′-GACAGAAACTTGAGCTGCATGGTGTGGGACAACAAGAAGG-3′)

The expression cassette was PCR amplified from TOPO Blunt/Pegl1-Fv43Dwith primers SK1236 and SK1297 to generate product for transformation ofT. reesei, using the electroporation method as described inWO2008153712A2.

15.5 Construction of the α-arabinofuranosidase Expression Cassette

For the construction of the F. verticilloides α-arabinofuranosidase genefv51A expression cassette, the fv51A gene product was amplified fromF.verticilloides genomic DNA using the primers SK1159 and SK1289. Aregion of the promoter of the endoglucanase gene egl1 was PCR amplifiedfrom T. reesei genomic DNA sample extracted from strain RL-P37, usingthe primers SK1236 and SK1262. These two PCR amplified DNA fragmentswere subsequently fused together in a fusion PCR reaction using theprimers SK1236 and SK1289. The resulting fusion PCR fragment was clonedinto pCR-Blunt II-TOPO vector (Invitrogen) to give the plasmid TOPOBlunt/Pegl1-Fv51A (FIG. 46) andE. coli One Shot® TOP10 ChemicallyCompetent cells (Invitrogen) were transformed using this plasmid.

Forward Primer SK1159: (SEQ ID NO: 107)(5′-CACCATGGTTCGCTTCAGTTCAATCCTAG-3′) Reverse Primer SK1289:(SEQ ID NO: 108) (5′-GTGGCTAGAAGATATCCAACAC-3′) Forward Primer SK1236:(SEQ ID NO: 109) (5′-CATGCGATCGCGACGTTTTGGTCAGGTCG-3′)Reverse Primer SK1262: (SEQ ID NO: 110)(5′-GAACTGAAGCGAACCATGGTGTGGGACAACAAGAAGGAC-3′)

The expression cassette was PCR amplified with primers SK1298 and SK1289to generate product for transformation of T. reesei using theelectroporation method.

Forward Primer SK1298:  (SEQ ID NO: 111) (5′-GTAGTTATGCGCATGCTAGAC-3′)Reverse Primer SK1289: (SEQ ID NO: 112) (5′-GTGGCTAGAAGATATCCAACAC-3′)

15.6 Co-Transformation of T. reesei with the β-glucosidase andendoxylanase Expression Cassettes

A Trichoderma reesei mutant strain, derived from RL-P37 (Sheir-Neiss, Get al. Appl. Microbiol. Biotechnol. 1984, 20:46-53) and selected forhigh cellulase production was co-transformed with the β-glucosidaseexpression cassette (cbhl promoter, T.reesei β-glucosidasel gene, cbh1terminator, and amdS marker), and the endoxylanase expression cassette(cbh1 promoter, T.reesei xyn3, and cbhl terminator) using PEG-mediatedtransformation (Penttila, M et al. Gene 1987, 61(2):155-64). Numeroustransformants were isolated and examined for β-glucosidase andendoxylanase production. One transformant called T. reesei strain #229was used for transformation with the other expression cassettes.

15.7 Co-transformation of T. reesei Strain #229 with two β-xylosidaseand α-arabinofuranosidase Expression Cassettes

T. reesei strain #229 was co-transformed with the β-xylosidase fv3Aexpression cassette (cbhl promoter, fv3A gene, cbh1 terminator, and alsRmarker), the β-xylosidase fv43D expression cassette (egl1 promoter,fv43D gene, native fv43D terminator), and the fv51Aα-arabinofuranosidase expression cassette (egl1 promoter, fv51A gene,fv51A native terminator) using electroporation. Transformants wereselected on Vogels agar plates containing chlorimuron ethyl (80 ppm).Vogels agar was prepared as follows, per liter.

50 x Vogels Stock Solution (below) 20 mL BBL Agar 20 g With deionizedH₂O bring to post-sterile addition: 980 mL 50% Glucose 20 mL 50 x VogelsStock Solution (WO 2005/001036), per liter: In 750 mL deionized H2O,dissolve successively: Na₃Citrate*2H₂O 125.00 g KH₂PO₄ (Anhydrous)250.00 g NH₄NO₃ (Anhydrous) 100 g MgSO₄*7H₂O 10.00 g CaCl₂*2H₂O 5.00 gVogels Trace Element Solution 5.0 mL Vogels Biotin Solution 2.5 mL Withdeionized H₂O, bring to 1 L

Numerous transformants were isolated and examined for β- xylosidase andL-α-arabinofuranosidase production. Transformants were also screened forbiomass conversion performance according to the cob saccharificationassay described in Example 1. Examples of T. reesei integratedexpression strains described herein are H3A, 39A, Al OA, 11A, and G9A,which express all of the genes for T. reesei beta-glucosidase 1, T.reesei Xyn3, Fv3A, Fv51A, and Fv43D, at different ratios (FIG. 78).Examples of T. reesei integrated expression strains described hereinalso include 44A, 69A, G6A and 102, and each includes most of the genesfor T. reesei beta-glucosidase 1, T. reesei XYN3, Fv3A, Fv51A, andFv43D, expressed at different ratios. Strain 44A lacked overexpressed T.reesei XYN3; strain 69A lacked Fv51A (confirmed by Western Blot, notshown); strains G6A and 102 lacked Fv3A (FIG. 78), as determined by HPLCprotein analysis (Example 1).

16. EXAMPLE 10: SACCHARIFICATION PERFORMANCE OF T. REESEI INTEGRATEDEXPRESSION STRAINS ON AMMONIA PRETREATED CORNCOB

The saccharification performance of enzyme compositions produced by T.reesei integrated expression strains on dilute ammonia pretreatedcorncob was evaluated. T. reesei enzyme samples were generated as eitherultrafiltration concentrates (UFC) or centrate. For the generation ofUFC, T. reesei fermentation broths (14L-scale) were obtained after cellseparation by centrifugation, concentrated usingmembrane-ultrafiltration through a Millipore 10 kD molecular weight cutoff membrane. Then pH was adjusted to 4.8. The cell-separated broth wasthen polished by filtration, using FW6 Buchner filtration. Each enzymesample was assayed for total protein concentration using the modifiedBiuret method.

The saccharification performance was evaluated in vials or in shakeflasks.

Each enzyme preparation was assayed for saccharification performance on20% dry solids (DS) loading of dilute ammonia pretreated corncob (see,WO2006/110901). All saccharification reactions were then titrated withsulfuric acid to pH 5.0 and sodium azide was added to a finalconcentration of 0.01% (w/v), for microbial contamination control. Eachsaccharification reaction was then dosed with 20 mg of total protein(TP) enzyme preparation per g of substrate glucan or xylan, asappropriate. Accellerase® 1500 (Ac1500) and the integrated strain UFC'swere dosed at 20 mg total protein/g glucan. An enzyme blend was preparedin a ratio of 25:9:4:3:1 Accellerase® 1500:Xyn3:Fv3A:Fv51A: FV43D. Inthe blend, Accellerase® 1500 was dosed at 20 mg total protein/g glucanand the hemicellulases were dosed per g xylan (4.3 mg Xyn3/g of xylan,1.7 mg Fv3A/g of xylan, 1.4 mg Fv51A/g of xylan, 0.5 mg Fv43D/g ofxylan). Each saccharification reaction was incubated at 50° C. in arotary shaker set to 200 rpm, then sampled and diluted 10 × (v/v) beforemonomeric sugar concentration was determined using HPLC analysis(detailed in Section 16.1 below under the “monomeric HPLC analysis”section) after 1, 2, 3 and 7 days of saccharification (FIGS. 47A, 47B).On day 3 of saccharification, each reaction was also sampled by weight(w/w) for oligomeric sugar concentration by HPLC analysis (Section 16.1,FIG. 47C). These results show that the enzyme compositions produced byintegrated strains H3A and G9A provide better glucose and xylose yieldsthan Accellerase®1500 or enzyme blends created from individuallyexpressed enzymes.

A chromatographic comparison of the enzyme composition produced by threedifferent integrated strains is shown in FIG. 47D.

16.1 HPLC Analysis

Monomeric Sugar HPLC Analysis: Each sample was analyzed by HPLC using aBioRad Aminex HPX-87H ion exclusion column (300 mm×7.8 mm). All day 1,2, 3, and 7 samples were diluted 10× volumetrically with 5 mM sulfuricacid, filtered through a 0.2 μm filter before injection into the HPLCand run under manufacture specifications. Oligomeric Sugar HPLC Analysis(acid hydrolysis): Day 3 saccharification samples were diluted 10× byweight with Milli-Q water, then sulfuric acid was added to the finalconcentration of 4% (w/w). A xylose and glucose standard (sugar recoverystandard—“SRS”) of known concentration was also prepared and measuredfor monomeric sugar HPLC analysis as stated above, and oligomer sugarHPLC analysis. Oligomer HPLC samples and were then autoclaved at 121° C.for 15 min, and filtered through a 0.2 μm filter before injection intothe HPLC BioRad Aminex HPX-87H ion exclusion column (300 mm x 7.8 mm),and run under manufacturer specifications.

Oligomer sugar concentration was determined by multiplying the percentretained xylose and glucose concentration of the sugar recovery standard(SRS) after acid hydrolysis, by the post acid hydrolysis HPLC sugarconcentrations of each sample, then subtracting the monomeric sugarconcentration determined in the “monomeric sugar HPLC Analysis” sectionabove.

17. EXAMPLE 11: IDENTIFICATION OF PHYLOGENETICALLY BROAD ENZYME CLASSESWHICH CAN IMPROVE THE SACCHARIFICATION PERFORMANCE OF T. REESEIINTEGRATED EXPRESSION STRAIN

In this example, enzyme activities which limit the efficacy of enzymecompositions produced by a T. reesei integrated expression strain wereidentified and enzymes from different species which can compensate forthose limiting activities or which could substitute for integratedstrain components are exemplified.

0.95g of pretreated corncob as described in Example 1 was added to 20 mLglass vials. Sufficient pH 5.0, 50 mM Sodium Acetate buffer and 1 NH₂SO₄ were added to give a total reaction weight of 3.00 g at 22 g drycorncob solids/100g of total slurry, pH 5.0 post enzyme additions. TheT. reesei enzyme composition produced by integrated strain H3A (FIGS.48A, 49A, and 50A) was added as an ultrafiltered (i.e., cell-free)concentrate to 7 mg total protein per g of glucan and xylan combined inthe feedstock to all vials. In addition, candidate hemicellulases wereadded as the ultrafiltered preparations from 14 L fermentation culturesexpressed by T. reesei Quad delete strain (described in WO 05/001036).The candidate hemicellulases were added at 0, 0.5, 1.0 or 3.0 mg enzymeprotein per g of glucan and xylan combined. The hemicellulases includedconstituents of the enzyme composition produced by the integrated strainitself including:

Fusarium verticillioides Fv3A

Fusarium verticillioides Fv51A

Fusarium verticillioides Fv43D

As well as enzymes from different fungi

Fusarium oxysporum Fo43A

Gibberella zeae Gz43A

Penicillium funiculosum Pf43A

Aspergillus fumigatus Af43A

Podospora anserina Pa51A

Penicillium funiculosum Pf51A

The vials were incubated at 180 rpm in an orbital shaker at 48° C. for72 h. Then 12 mL of water was added. A sub-sample was taken and furtherdiluted, centrifuged and filtered for HPLC analysis for monomer sugarsas described in Example 1.

The results shown in FIG. 48A and FIG. 48B demonstrate that addition ofβ-xylosidase activities such as Fusarium verticillioides Fv43D andFusarium oxysporum Fo43A markedly improved monomer xylose releaserelative to 7 mg T. reesei integrated strain total protein per g ofGlucan and Xylan combined. Significant improvements were even observedat the low additions of 0.5 and 1.0 mg/g.

The results shown in FIG. 49A and FIG. 49B demonstrate that addition ofall of the hemicellulases lead to a significant increase in monomerglucose, even >10% at hemicellulase loadings of 1-3 mg per g of Glucanand Xylan combined.

The results shown in FIG. 50A and FIG. 50B demonstrate that addition ofGH51 enzymes such as Fusarium verticillioides Fv51A, and especiallyPodospora anserine Pa51A and Penicillium funiculosum Pfu51A, led to anincrease in monomer arabinose level.

18. EXAMPLE 12: SACCARIFICATION OF VARIOUSLY PRETREATED SWITCHGRASS BYCELLULASE AND HEMICELLULASE PREPARATIONS

The saccharification performance of expressed cellulases andhemicellulases on pretreated raw switchgrass was evaluated. A range ofconditions for dilute ammonia pretreatment of switchgrass were evaluatedfor saccharification performance with an enzyme cocktail composed ofenzymes described herein. Pretreatment conditions vary and pretreatmentefficacy affects enzymatic hydrolysis performance.

Pretreatment of raw switchgrass was performed in sealed, 6″×½, stainlesssteel tubes that were immersed in a heated sand bath. A slurry of rawswitchgrass, water, and ammonium hydroxide (˜28% solution) was mixed tothe desired percent solids and percent ammonia and then loaded into apretreatment tube. Tubes were then held at the desired temperature(+/−2° C.) for the desired time and then quenched in ice water forapproximately 1 min before being brought to room temperature. Thepretreated slurry was removed from the tubes and allowed to dryovernight in the hood (>90% solids attainable).

Dried pretreated solids were then saccharified at 10% solids, pH 5, 50°C., 200 rpm using Accellerase® 1500, Xyn 3, Fv3A, Fv51A, and Fv43D (25,9, 7, 3, 1 mg total protein/g glucan or xylan respectively). A 5 mLtotal hydrolysate volume in 20 mL scintillation vials was used.

Glucan and xylan yields were based on monomeric glucose and xylosereleased compared to the glucan or xylan available from the raw biomass.Monomeric sugar concentrations were measured by HPLC (BioRad Aminex HPX87-H column).

Pretreatment time, temperature, percent solids, and percent NH₃, werevaried over a wide range in order to optimize saccharification results.Each pretreatment condition that had both glucan and xylan yields betterthan ˜50% is considered a strong performer. The pretreatment parametersthat performed strongly are listed in FIG. 79 along with theirrespective glucan, xylan, and total percent yields. Glucan and Xylanconversions are based on monomeric sugars released duringsaccharification as compared to glucan or xylan theoretically availablein the raw switchgrass. Total conversion is glucose and xylose only(FIG. 51).

19. EXAMPLE 13: SACCHARIFICATION OF PRETREATED SWITCHGRASS BY CELLULASEAND HEMICELLULASE PREPARATIONS

In addition to the above Examples, the saccharification performance ofenzyme mixes and enzyme compositions produced by an integrated strainwas tested on several substrates, pretreatments and conditions. Theseexperiments show the range of performance using the enzyme mix or anintegrated strain product. They demonstrate good performance across arange of substrates and pretreatments, pH, and temperatures.

Dilute ammonia pretreated switchgrass was prepared according to themethods and process ranges in WO06110901A: Switchgrass (38.7% glucan,22.2% xylan, 2.5% arabinan, 23.2% lignin) was hammer-milled to passthrough a 1 mm screen, then pretreated at 160° C. for 90 min with 6% NH₃(weight / weight DM, added as NH₄OH). This pretreated substrate wastreated with enzyme mixes containing Accellerase® 1500, Multifect®Xylanase (both commercial products of Danisco A/S, Genencor Division,Palo Alto, Calif.), Fv3A, Fv51A, and Fv43D in a total reaction mass of50g at 15% solids. The total protein (TP) of the commercial products wasdetermined by Biuret assay. The other enzymes were ultra-filtrationconcentrates (UFCs) following expression in cellulase quad-deletedstrains of T. reesei, with TP determined by Total Nitrogen analysis ofTCA-precipitable protein. All reactions were dosed with Accellerase®1500 at 25 mg TP/g Glucan and Multifect® Xylanase (MF Xyl) at 9 mg TP/gXylan, and Fv3A, Fv51A, and Fv43D were added as indicated in FIGS.56A-56B at 3.6 mg TP/g Xylan , 3.0 mg TP/g Xylan and 1.0 mg TP/g Xylan,respectively. All enzymes were dosed relative to the startingcarbohydrate contents of the switchgrass before pretreatment. Thesaccharification reactions were carried out at 47° C. and 33° C. at pH5.3 for three days.

The results at 33° C. showed that addition of Fv3A, Fv51A, and Fv43Dincreased glucan conversion (FIG. 56A) and more than doubled the xylanconversion (FIG. 56B). The results at 47° C. showed that addition ofFv3A, Fv51A, and Fv43D gave some increased glucan conversion (FIG. 56A)and more than doubled the xylan conversion (FIG. 56B). Additions ofFv51A or Fv43D, alone, gave large increases in xylan conversion,especially to xylo-oligomers or xylose monomers, respectively. Additionof Fv3A alone increased xylose yields, but in combination with Fv51Agave a large increase in xylan conversion to monomer.

20. EXAMPLE 14: SACCHARIFICATION OF PRETREATED SWITCHGRASS BY ANINTEGRATED T. REESEI STRAIN

The saccharification performance of an enzyme composition produced by anintegrated T. reesei strain (H3A) was evaluated on dilute ammoniapretreated switchgrass prepared according to the methods and processranges in WO06110901A: Switchgrass (37% glucan, 21% xylan, 5% arabinan,18% lignin) was hammer-milled to pass through a 1 mm screen, thenpretreated at 160° C. for 90 min with 10.0% NH₃ (weight/weight DM, addedas NH₄OH). In duplicate 500 mL glass Erlenmeyer flasks, 50 g ofpretreated slurry at 25% solids was saccharified at 48° C., pH 5.3 for 7days, with supernatant from the integrated strain was dosed at 14 mgTP/g of carbohydrate (glucan plus xylan). TP of H3A was determined byTotal Nitrogen analysis of TCA-precipitable protein. Enzymes were dosedrelative to the starting carbohydrate contents of the switchgrass beforepretreatment.

At the end of 7 days, high levels of glucan conversion (52-55%, FIG.57A) and xylan conversion (51%-53%, (FIG. 57B) were measured by HPLC.These results show that the enzyme composition produced by theintegrated strain can saccharify dilute ammonia pretreated switchgrassat high solids (25% dry matter).

21. EXAMPLE 15: SACCHARIFICATION OF HARDWOOD PULP BY AN INTEGRATED T.REESEI STRAIN

The saccharification performance of an enzyme composition produced byintegrated T reesei strain H3A was evaluated on industrial hardwoodunbleached pulp (derived from Kraft process and oxygen delignification,Smurfit Kappa Cellulose Du Pin, Biganos, France) with the followingcomposition: Glucan 75.1%, Xylan 19.1%, Acid soluble lignin 2.2%. Theenzymatic saccharification studies were carried out using NREL standardassay method LAP-009 “Enzymatic Saccharification of LignocellulosicBiomass” (www.nrel.gov/biomass/pdfs/42629.pdf), except that thecellulose loading was different (varying from 9.3-20%) and a total massof 100 g was used. The experimental condition was 200 rpm and pH 5.0,50° C. Enzyme was dosed at 20 mg TP/g glucan (based on final dry matter)at the start of the experiment. Samples were taken at timed intervals ifthey were liquefied, and then analyzed by HPLC for sugar concentration.Glucose, xylose, and cellobiose concentration were determined using aWaters HPLC system (Alliance system, Waters Corp., Milford, Mass.). TheHPLC column used for sugar analysis was from BioRad (Aminex HPX-87H ionexclusion column (300 mm×7.8 mm), BioRad Inc., Hercules, Calif.). Allsamples were diluted 10× with 5 mM sulfuric acid, filtered through a 0.2μm filter before injection into the HPLC. As indicated (FIG. 80) twoexperiments were carried out in “fed-batch” mode: Pretreated hardwoodpulp to an initial dry matter content of 7.0% at Time 0 and the rest ofthe substrate was added at discrete times in four equal portions duringthe first 24 h to bring the final dry solids loading to 20%.

Results showed high levels of glucan and xylan conversion to monomers(up to 89% and 90%, respectively) with the enzyme composition producedby integrated strain H3A. Conversions increased with higher enzymeloadings and longer saccharification times. Conversion was lower whenthe solids were at 20% than at 15%, but this lower conversion at 20%could be partially mitigated by using a fed-batch process.

22. EXAMPLE 16: SACCHARIFICATION OF HARDWOOD PULP BY AN INTEGRATED T.REESEI STRAIN OVER A RANGE OF TEMPERATURE AND PH

The saccharification performance of an enzyme composition produced by anintegrated T. reesei strain (H3A) on industrial hardwood unbleached pulp(as used in the preceding example) at 7% cellulose loading and 20 mgTP/g glucan of integrated strain supernatant (based on final dry matterand composition of the pretreated substrate) was tested at temperaturesfrom 45° C.-60° C. and pH's from 4.65-5.4 (buffered in 0.1 M sodiumcitrate). Results after 2 days saccharification are shown in FIG. 81 andshow good glucan and xylan conversions over the whole range ofconditions tested. These results suggested optimum conditions forsaccharification of this substrate as pH 4.9 and 50° C. but goodconversions are seen even at pH 5.0, 60° C. In a follow-up experiment at50° C., including lower pH's (and otherwise unchanged experimentalconditions) good saccharifications were seen at pH 3.8, pH 4.0 and pH4.25, with glucose & xylose titers of 45.1 & 9.7 g/L; 50.0 & 11.4 g/L;and 57.2 & 13.2 g/L, respectively.

23. EXAMPLE 17: SACCHARIFICATION OF STEAM-EXPANDED SUGARCANE BAGASSEWITH AN ENZYME COMPOSITION PRODUCED BY AN INTEGRATED STRAIN AT DIFFERENTENZYME DOSES

The saccharification performance of an enzyme composition produced by anintegrated T reesei strain (H3A) on steam-expanded sugarcane bagasse wasevaluated at 7% cellulose loading. The bagasse was pretreated by steaminjection in a StakeTech reactor at 210 psig, 200° C. with a 4 minresidence time. The pretreated material had the following composition:Glucan 40.9%, Xylan 20.8%, Lignin 27%. The integrated strain supernatantwas dosed at 10, 20, 30, 50 or 80 mg TP/g glucan (based on final drymatter and composition of the pretreated substrate). Saccharificationwas carried out in a 5 mL reaction volume at 50° C., pH 5 for 3 days.The results (FIGS. 58A-C) showed that the integrated strain productout-performed Accellerase® 1500 (20 mg protein/g glucan) in glucanconversion to glucose (FIG. 58A and FIG. 58C) and, especially, xylanconversion to xylose (FIG. 58B and FIG. 58C) even at half theAccellerase® 1500 dose. Glucan conversion, especially at Day 1,increased significantly as the dose of H3A total protein increased (FIG.58A and FIG. 58C), whereas xylan conversion was more rapid, with littleincrease from Day 1 to Day 3, and very high, even at the lowest dose ofH3A total protein (FIG. 58B and FIG. 58C).

24. EXAMPLE 18: SACCHARIFICATION OF DILUTE-ACID PRETREATED CORN FIBERWITH VARIOUS ENZYMES

The saccharification performance of enzyme mixtures on dilute sulfuricacid pretreated corn fiber was evaluated in a 250 mL shake flask. In atypical experiment, corn fiber (initial composition 38% C6 sugars and27% C5 sugars) was adjusted to 15% DS (dry solids) and 0.36% (w/w%)sulfuric acid was added. Corn fiber slurry was then autoclaved at 121°C. for 60 min. The slurry was then adjusted to pH 5.0 using 6 N NaOH.The sugar content of the pretreated sample was 21 g/L glucose and 12 g/Lxylose. Enzymes were added to the pretreated substrate as follows (asindicated in FIGS. 59A, 59B, 59C); Accellerase® 1500 (AC 1500): 20 mgTP/g glucan and 45 mg TP/g glucan; Accellerase® 1500 +Multifect®Xylanase (MF): (25 mg/g glucan +9mg TP/g xylan) and (25 mg TP/gglucan+20 mg TP/g xylan); Accellerase® 1500 +Xyn 3+Fv3A+Fv51A +Fv43D:(25 mg TP/g glucan +9 mg TP/g xylan +7 mg TP/g xylan +3 mg TP/g xylan +1mg TP/g xylan) (the full enzyme “blend”). The total protein (TP) of thecommercial products (Accellerase® 1500 and Multifect® Xylanase: DaniscoUS Inc., Genencor) were determined by Biuret assay. The other enzymeswere ultra-filtration concentrates (UFCs) following expression incellulase quad-deleted strains of T. reesei (as described earlier)—theirTP were determined by Total Nitrogen analysis of TCA-precipitableprotein. All enzymes were dosed relative to the starting carbohydratecontents of the corn fiber before pretreatment. Enzymaticsaccharification was carried out at 200 rpm and 50° C. for 24 h, 48 hand 120 h. Samples were withdrawn at different time intervals andanalyzed for the formation of glucose and xylose sugars by HPLC. Theadjusted sugar (glucose or xylose) reflected the sugar being producedfrom the enzymatic step, with the starting sugars subtracted.

The results show that the full enzyme blend out-performed the(Accellerase® 1500+Multifect® Xylanase) (“AC 1500 +MF”) in glucanconversion and in xylan conversion, even when both were dosed at thesame total protein. The full enzyme blend gave almost complete glucanconversion of this substrate after 5 days saccharification.

25. SPECIFIC EMBODIMENTS AND INCORPORATION BY REFERENCE

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure(s).

What is claimed is:
 1. A non-naturally occurring composition comprising:a. a first polypeptide or a variant thereof having β-xylosidaseactivity, L-α-arabinofuranosidase activity, or both β-xylosidaseactivity and L-α-arabinofuranosidase activity; and b. a secondpolypeptide or a variant thereof having xylanase activity.
 2. Thecomposition of claim 1, wherein the first polypeptide or variant thereofis an Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fv43A, Fv43B, Pa51A, Fo43A,Gz43A, Trichoderma reesei Bxl1, Fv51A, Af43A, or Pf51A polypeptide. 3.The composition of claim 1 or 2, wherein the first polypeptide orvariant thereof has β-xylosidase activity.
 4. The composition of claim3, wherein the first polypeptide or variant thereof having β-xylosidaseactivity is an Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fv43A, Fv43B, Pa51A,Fo43A, Gz43A, or Trichoderma reesei Bxl1 polypeptide.
 5. The compositionof any one of claims 1-4, further comprising a third polypeptide or avariant thereof having L-α-arabinofuranosidase activity.
 6. Thecomposition of claim 5, wherein the third polypeptide or variant thereofis an Fv51A, Af43A, Fv43B, Pa51A or Pf51A polypeptide.
 7. Thecomposition of any one of claims 1-4, further comprising a thirdpolypeptide having β-xylosidase activity.
 8. The composition of claim 7,wherein the first polypeptide is an Fv3A polypeptide or an Fv43Apolypeptide, and the third polypeptide is an Fv43D, Pa51A, Gz43A,Trichoderma reesei Bxl1, Pf43A, Fv43E, Fv39A, Fo43A, or Fv43Bpolypeptide.
 9. The composition of any one of claims 2, 4, 6, and 8,wherein, if present, the Fv43D polypeptide has at least 90% sequenceidentity to SEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii)107-341, or (iv) 107-350 of SEQ ID NO:28.
 10. The composition of any oneof claims 2, 4, 6 and 8, wherein, if present, the Fv3A polypeptide hasat least 90% sequence identity to the amino acid sequence of SEQ IDNO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv)395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2.
 11. The compositionof any one of claims 2, 4, 6, and 8, wherein, if present, the Pf43Apolypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii)21-323, (iv) 21-444, (v) 302- 444; (vi) 302-445, (vii) 324-444, or(viii) 324-445 of SEQ ID NO:4.
 12. The composition of any one of claims2, 4, 6, and 8, wherein, if present, the Fv43E polypeptide has at least90% sequence identity to the amino acid sequence of SEQ ID NO:6, or toresidues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ IDNO:6.
 13. The composition of any one of claims 2, 4, 6, and 8, wherein,if present, the Fv39A polypeptide has at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:8, or to residues (i) 20-439, (ii)20-291, (iii) 145-291, or 145-439 of SEQ ID NO:8.
 14. The composition ofany one of claims 2, 4, 6, and 8, wherein, if present, the Fv43Apolypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii)23-320, (iv) 23-448, (v) 303-448, or (vi) 321-449 of SEQ ID NO:10. 15.The composition of any one of claims 2, 4, 6, and 8, wherein, ifpresent, the Fv43B polypeptide has at least 90% sequence identity to theamino acid sequence of SEQ ID NO:12, or to residues (i) 17-574, (ii)27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12.
 16. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Pa51A polypeptide has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:14, or to residues (i) 21-676, (ii) 21- 652,(iii) 469-652, or (iv) 469-676 of SEQ ID NO:14.
 17. The composition ofany one of claims 2, 4, 6, and 8, wherein, if present, the Fo43Apolypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii)21-348, or (iv) 107-348 of SEQ ID NO:18.
 18. The composition of any oneof claims 2, 4, 6, and 8, wherein, if present, the Gz43A polypeptide hasat least 90% sequence identity to the amino acid sequence of SEQ IDNO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv)53-383 of SEQ ID NO:16.
 20. The composition of any one of claims 2, 4,6, and 8, wherein, if present, the Trichoderma reesei Bxl1 polypeptidehas at least 90% sequence identity to the amino acid sequence of SEQ IDNO:44.
 21. The composition of any one of claims 2, 4, 6, and 8, wherein,if present, the Fv51A polypeptide has at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:32, or to residues (i) 21-660, (ii)21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32.
 22. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Af43A polypeptide has at least 90% sequence identity to SEQ IDNO:20, or to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20. 23.The composition of any one of claims 2, 4, 6, and 8, wherein, ifpresent, the Pf51A polypeptide has at least 90% sequence identity to theamino acid sequence of SEQ ID NO:22, or to residues (i) 21-632, (ii)461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22.
 24. Thecomposition of any one of claim 2, 4, 6, or 8, wherein, if present, theFv43D polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:27, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:27, or toa fragment thereof.
 25. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Fv3A polypeptide is encoded by a nucleicacid having at least 90% sequence identity to SEQ ID NO:1, or by anucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:1, or to a fragment thereof.
 26. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Pf43A polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:3, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:3, or toa fragment thereof.
 27. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Fv43E polypeptide is encoded by anucleic acid having at least 90% sequence identity to SEQ ID NO:5, or bya nucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:5, or to a fragment thereof.
 28. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Fv39A polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:7, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:7, or toa fragment thereof.
 29. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Fv43A polypeptide is encoded by anucleic acid having at least 90% sequence identity to SEQ ID NO:9, or bya nucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:9, or to a fragment thereof.
 30. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Fv43B polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:11, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:11, or toa fragment thereof.
 31. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Pa51A polypeptide is encoded by anucleic acid having at least 90% sequence identity to SEQ ID NO:13, orby a nucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:13, or to a fragment thereof.
 32. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Fo43A polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:17, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:17, or toa fragment thereof.
 33. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Gz43A polypeptide is encoded by anucleic acid having at least 90% sequence identity to SEQ ID NO:15, orby a nucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:15, or to a fragment thereof.
 34. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Fv51A polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:31, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:31, or toa fragment thereof.
 35. The composition of any one of claims 2, 4, 6,and 8, wherein, if present, the Af43A polypeptide is encoded by anucleic acid having at least 90% sequence identity to SEQ ID NO:19, orby a nucleic acid that is capable of hybridizing, under high stringencyconditions, to SEQ ID NO:19, or to a fragment thereof.
 36. Thecomposition of any one of claims 2, 4, 6, and 8, wherein, if present,the Pf51A polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:21, or by a nucleic acid that is capableof hybridizing, under high stringency conditions, to SEQ ID NO:21, or toa fragment thereof.
 37. The composition of any one of claims 1-36,wherein the second polypeptide is a Trichoderma xylanase or anAspergillus xylanase.
 38. The composition of claim 37, wherein theTrichoderma xylanase is a Trichoderma reesei Xyn3 polypeptide or aTrichoderma reesei Xyn2 polypeptide.
 39. The composition of claim 38,wherein, if present, the Trichoderma reesei Xyn3 polypeptide has atleast 90% sequence identity to SEQ ID NO:42, or to residues 17-347 ofSEQ ID NO:42.
 40. The composition of claim 38, wherein, if present, theTrichoderma reesei Xyn2 polypeptide has at least 90% sequence identityto SEQ ID NO:43, or to residues 33-222 of SEQ ID NO:43.
 41. Thecomposition of claim 38, wherein, if present, the Trichoderma reeseiXyn3 polypeptide is encoded by a nucleic acid having at least 90%sequence identity to SEQ ID NO:41, or by a nucleic acid that is capableof hybridizing under high stringency conditions to SEQ ID NO:41, or to afragment thereof.
 42. The composition of claim 37, wherein theAspergillus xylanase is an AfuXyn2 polypeptide or an AfuXyn5polypeptide.
 43. The composition of claim 42, wherein, if present, theAfuXyn2 polypeptide has at least 90% sequence identity to SEQ ID NO:24,or to residues 19-228 of SEQ ID NO:24.
 44. The composition of claim 42,wherein, if present, the AfuXyn5 polypeptide has at least 90% sequenceidentity to SEQ ID NO:26, or to residues 20-313 of SEQ ID NO:26.
 45. Thecomposition of claim 42, wherein, if present, the AfuXyn2 polypeptide isencoded by a nucleic acid having at least 90% sequence identity to SEQID NO:23, or by a nucleic acid that is capable of hybridizing under highstringency to SEQ ID NO:23, or to a fragment thereof.
 46. Thecomposition of claim 42, wherein, if present, the AfuXyn5 polypeptide isencoded by a nucleic acid having at least 90% sequence identity to SEQID NO:25, or by a nucleic acid that is capable of hybridizing understringent conditions to a complement of SEQ ID NO:25, or to a fragmentthereof.
 47. The composition of any one of claims 1-46, furthercomprising one or more polypeptides having cellulase activity.
 48. Thecomposition of claim 47, wherein the one or more polypeptides havingcellulase activity are independently components of a whole cellulase.49. The composition of claim 48, wherein the whole cellulase is aβ-glucosidase-enriched whole cellulase.
 50. The composition of claim 48or 49, wherein the whole cellulase is a filamentous fungal wholecellulase.
 51. The composition of any one of claims 48-50, wherein thefilamentous fungal whole cellulase is a preparation from an Acremonium,Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora,Neurospora, Penicillium, Scytalidium, Thielavia, Chrysosporium,Phanerochaete, Tolypocladium, or Trichoderma species.
 52. Thecomposition of any one of claims 48-50, wherein the whole cellulase is aChrysosporium lucknowense, Phanerochaete chrysosporium, Trichodermareesei, Trichoderma harzianum, Trichoderma Trichoderma longibrachiatum,Trichoderma viride, or Pencillium funiculosum whole cellulase.
 53. Thecomposition of claim 47, wherein the one or more polypeptides havingcellulase activity are selected from a β-glucosidase, an endoglucanase,and a cellobiohydrolase.
 54. The composition of claim 49 or 53, whereinthe β-glucosidase is a Trichoderma reesei Bgl1 polypeptide.
 55. Thecomposition of claim 54, wherein the Trichoderma reesei Bgl1 polypeptidehas at least 90% sequence identity to SEQ ID NO:45, or to residues32-744 of SEQ ID NO:45.
 56. The composition of any one of claims 49 and53-55, wherein the β-glucosidase polypeptide constitutes up to 50 wt. %of the total weight of cellulase enzymes in the composition.
 57. Thecomposition of any one of claims 49 and 53-56, wherein the β-glucosidasepolypeptide constitutes 2 wt. % to 50 wt. % of the total weight ofproteins in the composition.
 58. The composition of any one of claims47-57, wherein the one or more polypeptides having cellulase activityconstitute 30 wt. % to 80 wt. % of the total weight of proteins in thecomposition.
 59. The composition of claim 58, wherein the one or morepolypeptides having cellulase activity together are capable of achievingat least 0.00005 fraction product per mg protein per gram of phosphoricacid swollen cellulose (PASO) as determined by a calcofluor assay. 60.The composition of any one of claims 1-59, further comprising one ormore accessory proteins.
 61. The composition of claim 60, wherein theone or more accessory proteins are selected from the group consisting ofmannanases, galactanases, arabinases, ligninases, amyloases,glucuronidases, proteases, esterases, lipases, xyloglucanases, glycosidehydrolase Family 61 polypeptides, CIP1, CIP2, swollenin, and expansins.62. The composition of claim 60, wherein the one or more accessoryproteins are selected from the group consisting of CIP1-like proteins,CIP2-like proteins, cellobiose dehydrogenases, and manganeseperoxidases.
 63. The composition of any one of claims 60-62, wherein theone or more accessory proteins constitute 1 wt. % to 50 wt. % of thetotal weight of proteins in the composition.
 64. The composition of anyone of claims 1-63, capable of converting 60% or more of thehemicellulose xylan from a biomass into xylose.
 65. The composition ofclaim 64, capable of converting 70% or more of the hemicellulose xylanfrom a biomass into xylose.
 66. The composition of claim 65, capable ofconverting 80% or more of the hemicellulose xylan from a biomass intoxylose.
 67. The composition of any one of claims 64-66, wherein thebiomass is corncob, corn stover, corn fiber, switchgrass, sorghum,paper, pulp, or sugarcane bagasse.
 68. The composition of any one ofclaims 64-66, wherein the biomass is Miscanthus.
 69. The composition ofany one of claims 1-68, further comprising a biomass.
 70. Thecomposition of claim 69, wherein the combined weight of polypeptideshaving xylanase activity is 1 g to 40 g per 1 kg of hemicellulose in thebiomass.
 71. The composition of claim 69, wherein the combined weight ofpolypeptides having xylanase activity is 0.5 g to 40 g per 1 kg ofhemicellulose in the biomass.
 72. The composition of claim 71, whereinthe combined weight of polypetpides having xylanase activity is 0.5 g to20 g per 1 kg of hemicellulose in the biomass.
 73. The composition ofany one of claims 70-72, wherein the combined weight of polypeptideshaving xylanase activity is 2 g to 20 g per 1 kg of hemicellulose in thebiomass.
 74. The composition of any one of claims 69-73, wherein thecombined weight of polypeptides having β-xylosidase activity is 1 g to50 g per 1 kg of hemicellulose in the biomass.
 75. The composition ofany one of claims 69-73, wherein the combined weight of polypeptideshaving β-xylosidase activity is 0.5 g to 50 g per 1 kg of hemicellulosein the biomass.
 76. The composition of claim 75, wherein the combinedweight of polypeptides having β-xylosidase activity is 0.5 g to 40 g per1 kg of hemicellulose in the biomass.
 77. The composition of any one ofclaims 74-76, wherein the combined weight of polypeptides havingβ-xylosidase activity is 2 g to 40 g per 1 kg of hemicellulose in thebiomass.
 78. The composition of any one of claims 69-77, wherein thecombined weight of polypeptides having L-α-arabinofuranosidase activity,if present, is 0.5 g to 20 g per 1 kg of hemicellulose in the biomass.79. The composition of any one of claims 69-77, wherein the combinedweight of polypetpides having L-α-arabinofuranosidase activity, ifpresent, is 0.2 g to 20 g per 1 kg of hemicellulose in the biomass. 80.The composition of any one of claims 69-79, wherein the combined weightof polypeptides having cellulase activity, if present, is 1 g to 100 gper 1 kg of cellulose in the biomass.
 81. A fermentation brothcomprising the composition of any one of claims 1-80.
 82. Thefermentation broth of claim 81, which is the fermentation broth of afilamentous fungus.
 83. The fermentation broth of claim 82, wherein thefilamentous fungus is a Trichoderma, Humicola, Fusarium, Aspergillus,Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia,Mucor, Cochliobolus, Pyricularia, Phanerochaete, or Chrysosporium. 84.The fermentation broth of claim 82, wherein the filamentous fungus is aAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Neurospora intermedia, Penicilliumpurpurogenum, Penicillium canescens, Penicillium solitum, Penicilliumfuniculosum, Phanerochaete chrysosporium, Phiebia radiate, Pleurotuseryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa,Trametes versicolor, Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.85. The fermentation broth of claim 82, wherein the filamentous fungusis an Aspergillus, or a Fusarium.
 86. The fermentation broth of claim82, wherein the filamentous fungus is a Trichoderma reesei.
 87. Thefermentation broth of claim 82, wherein the filamentous fungus is aPenicillium funiculosum.
 88. The fermentation broth of any one of claims81-87, which is a cell-free fermentation broth.
 89. A method ofconverting biomass to sugars, comprising contacting the biomass with thecomposition of any one of claims 1-80, or with the fermentation broth ofany one of claims 81-88.
 90. A saccharification process comprisingtreating a material comprising hemicellulose with the composition of anyone of claims 1-80 or with the fermentation broth of any one of claims81-89.
 91. The process of claim 90, wherein the material comprisinghemicellulose is corncob, corn stover, corn fiber, switchgrass, sorghum,paper, pulp, or sugarcane bagasse.
 92. The process of claim 90, whereinthe material comprising hemicellulose is Miscanthus.
 93. The process ofany one of claims 90-92, which yields at least 60% xylose fromhemicellulose xylan of the material comprising hemicellulose.
 94. Theprocess of any one of claims 90-93, further comprising recoveringmonosaccharides.
 95. An isolated, synthetic, or recombiant nucleic acidencoding a polypeptide having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:28, or to the amino acid sequencecorresponding to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or(iv) 107-350 of SEQ ID NO:28, which has β-xylosidase activity.
 96. Anisolated, synthetic, or recombinant nucleic acid encoding a polypeptidehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:4, or to the amino acid sequence corresponding to residues (i)21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444, (vi)302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4, which hasβ-xylosidase activity.
 97. An isolated, synthetic, or recombinantnucleic acid encoding a polypeptide having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:6, or to the amino acidsequence corresponding to residues (i) 19-530, (ii) 29-530, (iii)19-300, or (iv) 29-300 of SEQ ID NO:6, which has β-xylosidase activity.98. An isolated, synthetic, or recombinant nucleic acid encoding apolypeptide having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:8, or to the amino acid sequence corresponding toresidues (i) 20-439, (ii) 20-291, (iii) 145-291, or (iv) 145-439 of SEQID NO:8, which has β-xylosidase activity.
 99. An isolated, synthetic, orrecombinant nucleic acid encoding a polypeptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:10, or to theamino acid sequence corresponding to residues (i) 23-449, (ii) 23-302,(iii) 23-320, (iv) 23-448, (v) 303-448, (vi) 303-449, (vii) 321-448, or(viii) 321-449 of SEQ ID NO:10, which has β-xylosidase activity.
 100. Anisolated, synthetic, or recombinant nucleic acid encoding a polypeptidehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:12, or to the amino acid sequence corresponding to residues (i)17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12, whichhas β-xylosidase and/or L-α-arabinofuranosidase activities.
 101. Anisolated, synthetic, or recombinant nucleic acid encoding a polypeptidehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:14, or to the amino acid sequence corresponding to residues (i)21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14,which has β-xylosidase and/or L-α-arabinofuranosidase activities. 102.An isolated, synthetic, or recombinant nucleic acid encoding apolypeptide having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:16, or to the amino acid sequence corresponding toresidues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ IDNO:16, which has β-xylosidase activity.
 103. An isolated, synthetic, orrecombinant nucleic acid encoding a polypeptide having at least 90%%sequence identity to the amino acid sequence of SEQ ID NO:18, or to theamino acid sequence corresponding to residues (i) 21-341, (ii) 107-341,(iii) 21-348, or (iv) 107-348 of SEQ ID NO:18, which has β-xylosidaseactivity.
 104. An isolated, synthetic, or recombinant nucleic acidencoding a polypeptide having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:20, or to the amino acid sequencecorresponding to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20,which has L-α-arabinofuranosidase activity.
 105. An isolated, synthetic,or recombinant nucleic acid encoding a polypeptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:22, or to theamino acid sequence corresponding to residues (i) 21-632, (ii) 461-632,(iii) 21-642, or (iv) 461-642 of SEQ ID NO:22, which hasL-α-arabinofuranosidase activity.
 106. An isolated, synthetic, orrecombinant nucleic acid encoding a polypeptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:2, or to theamino acid sequence corresponding to residues (i) 24-766, (ii) 73-321,(iii) 73-394, (iv) 395-622, (v) 24-622, or (iv) 73-622 of SEQ ID NO:2,which has β-xylosidase activity.
 107. An isolated, synthetic, orrecombinant nucleic acid encoding a polypeptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:32, or to theamino acid sequence corresponding to residues (i) 21-660, (ii) 21-645,(iii) 450-645, or (iv) 450-660 of SEQ ID NO:32, which hasL-α-arabinofuranosidase activity.
 108. An isolated polypeptide, whereinsaid polypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:28, or to the amino acid sequence corresponding toresidues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQID NO:28, which has β-xylosidase activity.
 109. An isolated polypeptide,wherein said polypeptide has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:4, or to the amino acid sequencecorresponding to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv)21-444, (v) 302-444, (vi) 302-445, (vii) 324-444, or (viii) 324-445 ofSEQ ID NO:4, which has β-xylosidase activity.
 110. An isolatedpolypeptide, wherein said polypeptide has at least 90% sequence identityto the amino acid sequence of SEQ ID NO:6, or to the amino acid sequencecorresponding to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv)29-300 of SEQ ID NO:6, which has β-xylosidase activity.
 111. An isolatedpolypeptide, wherein said polypeptide has at least 90% sequence identityto the amino acid sequence of SEQ ID NO:8, or to the amino acid sequencecorresponding to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or(iv) 145-439 of SEQ ID NO:8, which has β-xylosidase activity.
 112. Anisolated polypeptide, wherein said polypeptide has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:10, or to the aminoacid sequence corresponding to residues (i) 23-449, (ii) 23-302, (iii)23-320, (iv) 23-448, (v) 303-448, (vi) 303-449, (vii) 321-448, or (viii)321-449 of SEQ ID NO:10, which has β-xylosidase activity.
 113. Anisolated polypeptide, wherein said polypeptide has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:12, or to the aminoacid sequence corresponding to residues (i) 17-574, (ii) 27-574, (iii)17-303, or (iv) 27-303 of SEQ ID NO:12, which has β-xylosidase and/orL-α-arabinofuranosidase activities.
 114. An isolated polypeptide,wherein said polypeptide has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:14, or to the amino acid sequencecorresponding to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or(iv) 469-676 of SEQ ID NO:14, which has β-xylosidase and/orL-α-arabinofuranosidase activities.
 115. An isolated polypeptide,wherein said polypeptide has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:16, or to the amino acid sequencecorresponding to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv)53-383 of SEQ ID NO:16, which has β-xylosidase activity.
 116. Anisolated polypeptide, wherein said polypeptide has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:18, or to the aminoacid sequence corresponding to residues (i) 21-341, (ii) 107-341, (iii)21-348, or (iv) 107-348 of SEQ ID NO:18, which has β-xylosidaseactivity.
 117. An isolated polypeptide, wherein said polypeptide has atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:20,or to the amino acid sequence corresponding to residues (i) 15-558, or(ii) 15-295 of SEQ ID NO:20, which has L-α-arabinofuranosidase activity.118. An isolated polypeptide, wherein said polypeptide has at least 90%sequence identity to the amino acid sequence of SEQ ID NO:22, or to theamino acid sequence corresponding to residues (i) 21-632, (ii) 461-632,(iii) 21-642, or (iv) 461-642 of SEQ ID NO:22, which hasL-α-arabinofuranosidase activity.
 119. An isolated polypeptide, whereinsaid polypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2, or to the amino acid sequence corresponding toresidues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v)24-622, or (iv) 73-622 of SEQ ID NO:2, which has β-xylosidase activity.120. An isolated polypeptide, wherein said polypeptide has at least 90%sequence identity to the amino acid sequence of SEQ ID NO:32, or to theamino acid sequence corresponding to residues (i) 21-660, (ii) 21-645,(iii) 450-645, or (iv) 450-660 of SEQ ID NO:32, which hasL-α-arabinofuranosidase activity.
 121. A host cell engineered torecombinantly express a polypeptide of any one of claims 108-120. 122.The host cell of claim 121, which is a cell of a filamentous fungus.123. The host cell of claim 122, which is a cell of a Trichoderma,Humicola, Fusarium, Aspergillus, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus,Pyricularia, Phanerochaete, or Chrysosporium.
 124. The host cell ofclaim 122, which is a cell of a Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumlucknowense, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkanderaadusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsiscaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta,Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsissubvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum,Penicillium canescens, Penicillium solitum, Penicillium funiculosum,Phanerochaete chrysosporium, Phiebia radiate, Pleurotus eryngii,Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.
 125. Thehost cell of claim 122 or 123, which is a cell of a Trichoderma spp., aPencillium, an Aspergillus, or a Fusarium.
 126. The host cell of claim125, wherein: (a) the Trichoderma spp. is Trichoderma reesei; or (b) thePencillium is Pencillium funiculosum; or (c) the Aspergillus isAspergillus oryzae or Aspergillus nidulans; or (d) the Fusarium isFusarium verticillioides or Fusarium oxysporum.
 127. The host cell ofclaim 121, which is a cell of a bacterium.
 128. The host cell of claim127, wherein the bacterium is a Streptomyces, a Thermomonospora, aBacillus, or a Cellulomonas.
 129. A method of producing the compositionof any one of claims 1-80 or the fermentation broth of any one of claims81-88.
 130. A process of saccharification comprising treating a biomassmaterial comprising hemicellulose with the composition of any one ofclaims 1-80 or the fermentation broth of any one of claims 81-88. 131.The process of claim 130, wherein the biomass material further comprisescellulose.
 132. The process of claim 130 or 131, wherein the amount ofpolypeptides having xylanase activity is 1 g to 40 g per kg ofhemicellulose in the biomass material.
 133. The process of claim 130 or131, wherein the amount of polypeptides having xylanase activity is 0.5g to 40 g per kg of hemicellulose in the biomass material.
 135. Theprocess of any one of claims 130-133, wherein the amount of polypeptideshaving β-xylosidase activity is 1 g to 50 g per kg of hemicellulose inthe biomass material.
 136. The process of any one of claims 130-133,wherein the amount of polypeptide having β-xylosidase activity is 0.5 gto 50 g per kg of hemicellulose in the biomass material.
 137. Theprocess of any one of claims 130-136, wherein the amount of polypeptideshaving L-α-arabinofuranosidase activity is 0.5 g to 20 g per kg ofhemicellulose in the biomass material.
 138. The process of any one ofclaims 130-136, wherein the amount of polypeptides havingL-α-arabinofuranosidase activity is 0.2 g to 20 g per kg ofhemicellulose in the biomass material.
 139. The process of any one ofclaims 130-138, wherein the amount of polypeptides having cellulaseactivity is 1 g to 100 g per kg of cellulose in the biomass material.140. The process of any one of claims 130-139, wherein the amount ofpolypeptides having β-glucosidase activity is 50% or less of the totalweight of polypeptides having cellulase activity.